Antibodies that bind il-4 and/or il-13 and their uses

ABSTRACT

The present invention relates to novel humanized anti-IL-4 and IL-13 antibodies and fragments thereof and novel bispecific antibodies and fragments thereof that specifically bind to IL-4 and IL-13. The invention also includes uses of the antibodies to treat or prevent IL-4 and/or IL-13 mediated diseases or disorders, including allergic asthma and dermatitis.

FIELD OF THE INVENTION

The present invention relates to novel anti-IL-4 antibodies, anti-IL-13antibodies and bispecific anti-IL-4/anti-IL-13 antibodies and their usein the amelioration, treatment or prevention of diseases or disorders inmammals, including humans, resulting from improper IL-4 and/or IL-13activity or metabolism. An antibody of interest may block engagementand/or signaling of a ligand, such as IL-4 or IL-13, with a receptor orreceptor complex, such as IL-4Rα, IL-13Rα1 and IL-13Rα2. Prophylactic,immunotherapeutic and diagnostic compositions comprising the antibodiesof interest and their use in methods for preventing or treating diseasesin mammals, including humans, caused by inappropriate metabolism and/oractivity of lymphoid and non-lymphoid cells, including monocytes,fibroblasts and endothelial cells, are disclosed. Such diseases includeautoimmune deficiencies and diseases caused by or characterized byinflammation, such as allergic asthma and dermatitis.

BACKGROUND OF THE INVENTION

Interleukin-4 (IL-4) is a pleiotropic cytokine that has a broad spectrumof biological effects on lymphoid B and T cells, and many non-lymphoidcells including monocytes, endothelial cells and fibroblasts. Forexample, IL-4 stimulates the proliferation of several IL-2- andIL-3-dependent cell lines, induces the expression of class II majorhistocompatability complex molecules on resting B cells, and enhancesthe secretion of IgG4 and IgE by human B cells. IL-4 is associated witha Th2-type immune response, and is produced by and promotesdifferentiation of Th2 cells. IL-4 has been implicated in a number ofdisorders, such as allergy and asthma.

IL-13 is a recently identified (Minty, A. et al., Nature, 1993, 362,248-250, and McKenzie, A. N. et al., Proc. Natl. Acad. Sci. U.S.A, 1993,90, 3735-3739) cytokine of 112 amino acids secreted by the activated Tlymphocytes, the B lymphocytes and the mastocytes after activation.

By virtue of its numerous biological properties shared with IL-4, IL-13has been described as an IL-4-like cytokine. Its activities are indeedsimilar to those of IL-4 on the B cells (Defiance, T. et al., J. Exp.Med., 1994, 179, 135-143, Punnonen, J. et al., Proc. Natl. Acad. Sci.(USA), 1993, 90, 3730-3734, Fior, R. et al., Eur. Cytokine Network,1994, 5, 593-600), the monocytes (Muzio, M. R. F. et al., Blood, 1994,83, 1738-1743, De Waal Malefyt, R. et al., J. Immunol, 1993, 151,6370-6381, Doyle, A. et al., Eur. J. Immunol. 1994, 24, 1441-1445,Montaner, L. J. et al., J. Exp. Med., 1993, 178, 743-747, Sozzani, P. etal., J. Biol. Chem., 1995, 270, 5084-5088) and other non-haematopoieticcells (Herbert, J. M. et al., Febs Lett., 1993, 328, 268-270, andDerocq, J. M. et al., Febs Lett. 1994, 343, 32-36). On the other hand,contrary to IL-4, it does not exert a specific effect on resting oractivated T cells (Zurawuki, G. et al., Immunol. Today, 1994, 15,19-26).

Various biological activities of IL-13 on the monocytes/macrophages, theB lymphocytes and certain haematopoietic precursors have been describedin detail by A. J. Minty as well as in review articles on IL-13. Severaldata indicate, in addition, that this cytokine has a pleiotropic effecton other cell types. These non-haematopoietic cells which are directlyaffected by IL-13 are endothelial and microglial cells, keratinocytesand kidney and colon carcinomas.

One of the stages in the analysis of the signal transmitted by abiological molecule within a cell consists in identifying its membranereceptor. The research studies carried out to this end on the IL-13receptor have shown that IL-13 and IL-4 have a common receptor, or atthe very least some of the components of a common receptor complex, aswell as common signal transduction elements (Zurawski S. M. et al., EmboJournal, 1993, 12, 2663-2670, Aversa, G. et al., J. Exp. Med., 1993,178, 2213-2218, Vita, N. et al., Biol. Chem., 1995, 270, 3512-3517,Lefort, S. et al., Febs Lett., 1995, 366, 122-126). This receptor ispresent at the surface of various cell types, in a variable numberaccording to the cell type considered. The comparative distribution ofthe IL-13 and IL-4 receptors has been indicated by A. J. Minty(Interleukin-13 for Cytokines in Health and Disease. Eds D. G. Remickand J. S. Frie, Marcel Decker, N.Y. 1996).

The cell surface receptors and receptor complexes bind IL-4 and/or IL-13with different affinities. The principle components of receptors andreceptor complexes that bind IL-4 and/or IL-13 are IL-4Rα, IL-13Rα1 andIL-13Rα2. These chains are expressed on the surface of cells as monomersor heterodimers of IL-4Rα/IL-13Rα1 (Type II IL-4R) or IL-4Rα/γc (Type IIL-4R). IL-4Rα monomer and IL-4Rα/γc heterodimer bind IL-4, but notIL-13. IL-13Rα1 and IL-13Rα2 monomers bind IL-13, but do not bind IL-4.IL-4Rα/IL-13Rα1 heterodimer binds both IL-4 and IL-13 (Murata et al.,Int. J. Hematol., 1999, 69, 13-20).

Th2-type immune responses promote antibody production and humoralimmunity, and are elaborated to fight off extracellular pathogens. Th2cells are mediators of Ig production (humoral immunity) and produceIL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 (Tanaka, et, al., CytokineRegulation of Humoral Immunity, 251-272, Snapper, ed., John Wiley andSons, New York (1996)). Th2-type immune responses are characterized bythe generation of certain cytokines (e.g., IL-4, IL-13) and specifictypes of antibodies (IgE, IgG4) and are typical of allergic reactions,which may result in watery eyes and asthmatic symptoms, such as airwayinflammation and contraction of airway muscle cells in the lungs.

Both IL-4 and IL-13 are therapeutically important cytokines based ontheir biological functions and play critical roles in many diseases,including asthma (Curr Opin Allergy Clin Immunol 2005, Vo. 5, 161-166).IL-4 has been shown to be able to inhibit autoimmune disease and IL-4and IL-13 have both shown the potential to enhance anti-tumor immuneresponses. Since both cytokines are involved in the pathogenesis ofallergic diseases, inhibitors of these cytokines could providetherapeutic benefits.

Accordingly, a need exists for improved agents that inhibit IL-4,inhibit IL-13, and single agents that inhibit both IL-4 and IL-13.

SUMMARY OF THE INVENTION

The present invention provides novel humanized monoclonal and bispecificantibodies, and fragments and derivatives thereof, which specificallybind to IL-4 and/or IL-13. Some of the anti-IL-4 and/or IL-13 mono- orbispecific antibodies, and fragments thereof, can be altered to preventintrachain disulfide bond formation resulting in a molecule that isstable through manufacturing and use in vivo. The antibodies of thepresent invention neutralize IL-4 and/or IL-13 activity in thebiological assays described herein.

The invention includes the amino acid sequences of the variable heavyand light chain of the antibodies and their corresponding nucleic acidsequences.

Another embodiment of the present invention includes the cell lines andvectors harboring the antibody sequences of the present invention.

Another embodiment of the present invention is the use of the antibodiesfor the preparation of a pharmaceutical composition for the treatment ofdiseases and disorders associated with IL-4 and/or IL-13 function andmetabolism. In particular, the present invention relates to thetreatment of cancer, autoimmune deficiencies and diseases caused by orcharacterized by inflammation, such as allergic asthma and dermatitis.

Additional features and advantages are described herein, and will beapparent from, the following Detailed Description and the figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a bispecific anti-IL-4/IL-13 antibodymolecule containing four polypeptide chains. Two lighter chains consistof N-VL_(hB-B13)-linker-VL_(h8D4-8)-CL-C (CL, light chain constantregion), two heavier chains consist ofN-VH_(hB-B13)-linker-VH_(h8D4-8)-CH1-CH2-CH3-C. The linker sequence(G4S)₂ is GGGGSGGGGS (SEQ ID NO: 6).

FIG. 2 illustrates the amino acid sequences of humanized variabledomains of B-B13 anti-IL-13 antibody (SEQ ID NOS: 1 and 2) and humanizedvariable domains of 8D4-8 anti-IL-4 antibody (SEQ ID NOS: 3, 4 and 5).Underline indicates amino acid changes made. Bold indicates the CDR.

DETAILED DESCRIPTION OF THE INVENTION

This invention is not limited to the particular methodology, protocols,cell lines, vectors, or reagents described herein because they may varywithout departing from the spirit and scope of the invention. Further,the terminology used herein is for the purpose of exemplifyingparticular embodiments only and is not intended to limit the scope ofthe present invention. Unless defined otherwise, all technical andscientific terms and any acronyms used herein have the same meanings ascommonly understood by one of ordinary skill in the art in the field ofthe invention. Any method and material similar or equivalent to thosedescribed herein can be used in the practice of the present inventionand only exemplary methods, devices, and materials are described herein.

All patents and publications mentioned herein are incorporated herein inentirety by reference for the purpose of describing and disclosing theproteins, enzymes, vectors, host cells and methodologies reportedtherein that might be used with and in the present invention. However,nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

Prior to teaching the making and using of the IL-4 and/or IL-13 relatedmethods and products of interest, the following non-limiting definitionsof some terms and phrases are provided to guide the artisan.

“Interleukin-4” (IL-4) relates to the naturally occurring, or endogenousmammalian IL-4 proteins and to proteins having an amino acid sequencewhich is the same as that of a naturally occurring or endogenouscorresponding mammalian IL-4 protein {e.g., recombinant proteins,synthetic proteins (i.e., produced using the methods of syntheticorganic chemistry)). Accordingly, as defined herein, the term includesmature IL-4 protein, polymorphic or allelic variants, and other isoformsof an IL-4 and modified or unmodified forms of the foregoing (e.g.,lipidated, glycosylated). Naturally occurring or endogenous IL-4includes wild type proteins such as mature IL-4, polymorphic or allelicvariants and other isoforms and mutant forms which occur naturally inmammals (e.g., humans, non-human primates). Such proteins can berecovered or isolated from a source which naturally produces IL-4, forexample. These proteins and proteins having the same amino acid sequenceas a naturally occurring or endogenous corresponding IL-4, are referredto by the name of the corresponding mammal. For example, where thecorresponding mammal is a human, the protein is designated as a humanIL-4. Several mutant IL-4 proteins are known in the art, such as thosedisclosed in WO 03/038041.

“Interleukin-13” (IL-13) refers to naturally occurring or endogenousmammalian IL-13 proteins and to proteins having an amino acid sequencewhich is the same as that of a naturally occurring or endogenouscorresponding mammalian IL-13 protein (e.g., recombinant proteins,synthetic proteins (i.e., produced using the methods of syntheticorganic chemistry)). Accordingly, as defined herein, the term includesmature IL-13 protein, polymorphic or allelic variants, and otherisoforms of IL-13 (e.g., produced by alternative splicing or othercellular processes), and modified or unmodified forms of the foregoing(e.g., Hpidated, glycosylated). Naturally occurring or endogenous IL-13include wild type proteins such as mature IL-13, polymorphic or allelicvariants and other isoforms and mutant forms which occur naturally inmammals (e.g., humans, non-human primates). For example, as used hereinIL-13 encompasses the human IL-13 variant in which Arg at position 110of mature human IL-13 is replaced with Gin (position 110 of mature IL-13corresponds to position 130 of the precursor protein) which is associedwith asthma (atopic and nonatopic asthma) and other variants of IL-13.(Heinzmann et al, Hum Mol Genet. 9:549-559 (2000).) Such proteins can berecovered or isolated from a source which naturally produces IL-13, forexample. These proteins and proteins having the same amino acid sequenceas a naturally occurring or endogenous corresponding IL-13 are referredto by the name of the corresponding mammal. For example, where thecorresponding mammal is a human, the protein is designated as a humanIL-13. Several mutant IL-13 proteins are known in the art, such as thosedisclosed in WO 03/035847.

The phrase “substantially identical” with respect to an antibody chainpolypeptide sequence may be construed as an antibody chain exhibiting atleast 70%, 80%, 90%, 95% or more sequence identity to the referencepolypeptide sequence. The term with respect to a nucleic acid sequencemay be construed as a sequence of nucleotides exhibiting at least about85%, 90%, 95%, or 97% or more sequence identity to the reference nucleicacid sequence.

The terms, “identity” or “homology” may mean the percentage ofnucleotide bases or amino acid residues in the candidate sequence thatare identical with the residue of a corresponding sequence to which itis compared, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent identity for the entiresequence, and not considering any conservative substitutions as part ofthe sequence identity. Neither N-terminal or C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are available and well known inthe art. Sequence identity may be measured using sequence analysissoftware.

The phrases and terms “functional fragment, variant, derivative oranalog” and the like, as well as forms thereof, of an antibody orantigen is a compound or molecule having qualitative biological activityin common with a full-length antibody or antigen of interest. Forexample, a functional fragment or analog of an anti-IL-4 antibody is onewhich can bind to an IL-4 molecule or one which can prevent orsubstantially reduce the ability of a ligand, or an agonistic orantagonistic antibody, to bind to IL-4.

“Substitutional” variants are those that have at least one amino acidresidue in a native sequence removed and replaced with a different aminoacid inserted in its place at the same position. The substitutions maybe single, where only one amino acid in the molecule is substituted, ormay be multiple, where two or more amino acids are substituted in thesame molecule. The plural substitutions may be at consecutive sites.Also, one amino acid can be replaced with plural residues, in which casesuch a variant comprises both a substitution and an insertion.“Insertional” variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative sequence. Immediately adjacent to an amino acid means connectedto either the α-carboxyl or α-amino functional group of the amino acid.“Deletional” variants are those with one or more amino acids in thenative amino acid sequence removed. Ordinarily, deletional variants willhave one or two amino acids deleted in a particular region of themolecule.

The term “antibody” is used in the broadest sense, and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), antibody fragments or synthetic polypeptidescarrying one or more CDR or CDR-derived sequences so long as thepolypeptides exhibit the desired biological activity. Antibodies (Abs)and immunoglobulins (Igs) are glycoproteins having the same structuralcharacteristics. Generally, antibodies are considered Igs with a definedor recognized specificity. Thus, while antibodies exhibit bindingspecificity to a specific target, immunoglobulins include bothantibodies and other antibody-like molecules which lack targetspecificity. The antibodies of the invention can be of any class (e.g.,IgG, IgE, IgM, IgD, IgA and so on), or subclass (e.g., IgG₁, IgG₂, IgG₂,IgG₃, IgG₄, IgA₁, IgA₂ and so on) (“type” and “class”, and “subtype” and““subclass”, are used interchangeably herein). Native or wildtype, thatis, obtained from a non-artificially manipulated member of a population,antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at the other end. By “non-artificially manipulated” ismeant not treated to contain or express a foreign antigen bindingmolecule. Wildtype can refer to the most prevalent allele or speciesfound in a population or to the antibody obtained from a non-manipulatedanimal, as compared to an allele or polymorphism, or a variant orderivative obtained by a form of manipulation, such as mutagenesis, useof recombinant methods and so on to change an amino acid of theantigen-binding molecule.

As used herein, “anti-IL-4 antibody” means an antibody or polypeptidederived therefrom (a derivative) which binds specifically to IL-4 asdefined herein, including, but not limited to, molecules which inhibitor substantially reduce the binding of IL-4 to its receptor or inhibitIL-4 activity.

As used herein, “anti-IL-13 antibody” means an antibody or polypeptidederived therefrom (a derivative) which binds specifically to IL-13 asdefined herein, including, but not limited to, molecules which inhibitor substantially reduce the binding of IL-13 to its receptor or inhibitIL-13 activity.

The term “variable” in the context of a variable domain of antibodiesrefers to certain portions of the pertinent molecule which differextensively in sequence between and among antibodies and are used in thespecific recognition and binding of a particular antibody for itsparticular target. However, the variability is not evenly distributedthrough the variable domains of antibodies. The variability isconcentrated in three segments called complementarity determiningregions (CDRs; i.e., CDR1, CDR2, and CDR3) also known as hypervariableregions, both in the light chain and the heavy chain variable domains.The more highly conserved portions of variable domains are called theframework (FR) regions or sequences. The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together often in proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the target (epitope or determinant) binding site ofantibodies (see Kabat et al. Sequences of Proteins of ImmunologicalInterest, National Institute of Health, Bethesda, Md. (1987)). As usedherein, numbering of immunoglobulin amino acid residues is doneaccording to the immunoglobulin amino acid residue numbering system ofKabat et al., unless otherwise indicated. One CDR can carry the abilityto bind specifically to the cognate epitope.

The term “hinge” or “hinge region” as used in the present inventionrefers to the flexible polypeptide comprising the amino acids betweenthe first and second constant domains of an antibody.

The term “antibody fragment” refers to a portion of an intact or afull-length chain or an antibody, generally the target binding orvariable region. Examples of antibody fragments include, but are notlimited to, F_(ab), F_(ab′), F_((ab′)2) and F_(v) fragments. A“functional fragment” or “analog of an anti-IL-4 and/or IL-13 antibody”is one which can prevent or substantially reduce the ability of thereceptor to bind to a ligand or to initiate signaling. As used herein,functional fragment generally is synonymous with, “antibody fragment”and with respect to antibodies, can refer to fragments, such as F_(v),F_(ab), F_((ab′)2) and so on which can prevent or substantially reducethe ability of the receptor to bind to a ligand or to initiatesignaling. An “F,” fragment consists of a dimer of one heavy and onelight chain variable domain in a non-covalent association (V_(H)-V_(L)dimer). In that configuration, the three CDRs of each variable domaininteract to define a target binding site on the surface of theV_(H)-V_(L) dimer, as in an intact antibody. Collectively, the six CDRsconfer target binding specificity on the intact antibody. However, evena single variable domain (or half of an F_(v) comprising only three CDRsspecific for a target) can have the ability to recognize and to bindtarget.

“Single-chain F_(v),” “sF_(v)” or “scAb” antibody fragments comprise theV_(H) and V_(L) domains of an antibody, wherein these domains arepresent in a single polypeptide chain. Generally, the F_(v) polypeptidefurther comprises a polypeptide linker, often a flexible molecule,between the V_(H) and V_(L) domains, which enables the sFv to form thedesired structure for target binding.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments can comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain. By using a linker that is tooshort to allow pairing between the two variable domains on the samechain, the diabody domains are forced to pair with the binding domainsof another chain to create two antigen-binding sites.

The F_(ab) fragment contains the variable and constant domains of thelight chain and the variable and first constant domain (C_(H1)) of theheavy chain. F_(ab′) fragments differ from F_(ab) fragments by theaddition of a few residues at the carboxyl terminus of the C_(H1) domainto include one or more cysteines from the antibody hinge region. F_(ab′)fragments can be produced by cleavage of the disulfide bond at the hingecysteines of the F_((ab′)2) pepsin digestion product. Additionalenzymatic and chemical treatments of antibodies can yield otherfunctional fragments of interest.

The term “linear Fab” refers to a tetravalent antibody as described byMiller et al. (2003), J. Immunol. 170: 4854-4861. The “linear Fab” iscomposed of a tandem of the same CH1-VH domain, paired with theidentical light chain at each CH1-VH position. These molecules have beendeveloped in order to increase the valency of an antibody to enhance itsfunctional affinity through the avidity effect, but they aremonospecific.

The term “bispecific antibodies (BsAbs)” refers to molecules whichcombine the antigen-binding sites of two antibodies within a singlemolecule. Thus, a bispecific antibody is able to bind two differentantigens simultaneously. Besides applications for diagnostic purposes,BsAbs pave the way for new therapeutic applications by redirectingpotent effector systems to diseased areas or by increasing neutralizingor stimulating activities of antibodies.

Initial attempts to couple the binding specificities of two wholeantibodies against different target antigens for therapeutic purposesutilized chemically fused heteroconjugate molecules (Staerz et al.(1985), Nature 314: 628-631).

Bispecific antibodies have been produced from hybrid hybridomas byheterohybridoma techniques and have demonstrated in vitro propertiessimilar to those observed for heteroconjugates (Milstein & Cuello (1983)Nature 305:537-540).

Despite the promising results obtained using heteroconjugates orbispecific antibodies produced from cell fusions as cited above, severalfactors made them impractical for large scale therapeutic applications.Such factors include: rapid clearance of large heteroconjugates in vivo,the labor intensive techniques required for generating either type ofmolecule, the need for extensive purification of heteroconjugates awayfrom homoconjugates or mono-specific antibodies and generally lowyields.

Genetic engineering has been used with increasing frequency to design,modify, and produce antibodies or antibody derivatives with a desiredset of binding properties and effector functions.

A variety of recombinant methods have been developed for efficientproduction of BsAbs, both as antibody fragments (Carter et al. (1995),J. Hematotherapy 4: 463-470; Pluckthun et al. (1997) Immunotechology 3:83-105; Todorovska et al. (2001) J. Immunol. Methods 248: 47-66) andfull length IgG formats (Carter (2001) J. Immunol. Methods 248: 7-15).

Combining two different scFvs results in BsAb formats with minimalmolecular mass, termed sc-BsAbs or Ta-scFvs (Mack et al. (1995), Proc.Acad. Sci. USA. 92: 7021-7025; Mallender et al. (1994) J. Biol. Chem.269: 199-206). BsAbs have been constructed by genetically fusing twoscFvs via dimerization functionality such as a leucine zipper (Kostelnyet al. (1992) J. Immunol. 148: 1547-53; de Kruif et al. (1996) J. Biol.Chem. 271: 7630-4).

As mentioned above, diabodies are small bivalent and bispecific antibodyfragments. The fragments comprise a VH connected to a VL on the samepolypeptide chain, by using a linker that is too short (less than 12amino acids) to allow pairing between the two domains on the same chain.The domains are forced to pair intermolecularly with the complementarydomains of another chain and create two antigen-binding sites. Thesedimeric antibody fragments, or “diabodies,” are bivalent and bispecific.(Holliger et al. (1993), Proc. Natl. Acad. Sci. USA. 90: 6444-6448).Diabodies are similar in size to a Fab fragment. Polypeptide chains ofVH and VL domains joined with linker between 3 and 12 amino acids formpredominantly dimers (diabodies), whereas with linker between 0 and 2amino acid residues, trimers (triabodies) and tetramers (tetrabodies)find favor. In addition to the linker length, the exact pattern ofoligomerization seems to depend on the composition as well as theorientation of the V-domains (Hudson et al. (1999), J Immunol Methods231: 177-189). The predictability of the final structure of diabodymolecules is very poor.

Although sc-BsAbs and diabodies based constructs display interestingclinical potential, it was shown that such non-covalently associatedmolecules are not sufficient stable under physiological conditions. Theoverall stability of a scFv fragment depends on the intrinsic stabilityof the VL and VH domains as well as on the stability of the domaininterface. Insufficient stability of the VH-VL interface of scFvfragments has often been suggested as a main cause of irreversible scFvinactivation, since transient opening of the interface, which would beallowed by the peptide linker, exposes hydrophobic patches that favoraggregation and therefore instability and poor production yield (Wornand Plückthun (2001), J. Mol. Biol. 305: 989-1010).

An alternative method of manufacturing bispecific bivalentantigen-binding proteins from VH and VL domains is disclosed in U.S.Pat. No. 5,989,830. Such double head antibody fragments are obtained byexpressing a dicistronic vector which encodes two polypeptide chains,whereby one polypeptide chain has two times a VH in series by a peptidelinker (VH1-linker-VH2) and the other polypeptide chain consisting ofcomplementary VL domains connected in series by a peptide linker(VL1-linker-VL2). It was described in U.S. Pat. No. 5,989,830 that eachlinker should comprise at least 10 amino acid residues.

Polyvalent protein complexes (PPC) with an increased valency aredescribed in US 2005/0003403 A1. PPCs comprise two polypeptide chainsgenerally arranged laterally to one another. Each polypeptide chaintypically comprises 3 or 4 “v-regions”, which comprise amino acidsequences capable of forming an antigen binding site when matched with acorresponding v-region on the opposite polypeptide chain. Up to about 6“v-regions” can be used on each polypeptide chain. The v-regions of eachpolypeptide chain are connected linearly to one another and may beconnected by interspersed linking regions. When arranged in the form ofthe PPC, the v-regions on each polypeptide chain form individual antigenbinding sites. The complex may contain one or several bindingspecificities.

However, the use of such molecules showed aggregation, unstability andpoor expression yield (Wu et al. (2001) Prot. Eng. 14: 1025-1033). Theseare typical stability problems that may occur expressing single chainbased antibodies. (Worn and Plückthun (2001), J. Mol. Biol. 305:989-1010).

Thus, it is the object of the present invention to provide a bispecificpolyvalent antibody by means of which the formation of aggregates can beavoided. Furthermore, it shall have a stability which makes it usablefor therapeutic uses.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts.

Monoclonal antibodies herein specifically include “chimeric” antibodiesin which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass (type or subtype), with the remainder of the chain(s) identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity of binding to IL-4 and/or IL-13 or impactingIL-4 and/or IL-13 activity or metabolism (U.S. Pat. No. 4,816,567; andMorrison et al., Proc Natl Acad Sci USA 81:6851 (1984)). Thus, CDRs fromone class of antibody can be grafted into the FR of an antibody ofdifferent class or subclass.

Monoclonal antibodies are highly specific, being directed against asingle target site, epitope or determinant. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes)of an antigen, each monoclonal antibody is directed against a singledeterminant on the target. In addition to their specificity, monoclonalantibodies are advantageous being synthesized by a host cell,uncontaminated by other immunoglobulins, and provides for cloning therelevant gene and mRNA encoding the antibody of chains thereof. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies for use withthe present invention may be isolated from phage antibody librariesusing well known techniques or can be purified from a polyclonal prep.The parent monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method described byKohler et al., Nature 256:495 (1975), or may be made by recombinantmethods well known in the art.

The term “polyvalent antibody” as used in the present invention refersto an antibody comprising two or more antigen binding sites, thus beingable to bind two or more antigens, which may have the same or adifferent structure, simultaneously. The term “bivalent” means that theantibody comprises two antigen binding sites. The term “tetravalent”means that the antibody comprises four antigen binding sites.

The term “antigen binding site” as used in the present invention refersto the part of the antibody which comprises the area which specificallybinds to and is complementary to part or all of an antigen. Where anantigen is large, an antibody may only bind to a particular part of theantigen, which part is termed on epitope. An antigen binding domain maybe provided by one or more antibody variable domains. Preferably, anantigen binding domain is made of the association of an antibody lightchain variable domain (VL) and an antibody heavy chain variable domain(VH).

The term “antigen” as used in the present invention refers to a moleculeor a portion of a molecule capable of being bound by the antibodies ofthe present invention. An antigen can have one or more than one epitope.Examples of antigens recognized by the antibodies of the presentinvention include, but are not limited to, serum proteins, e.g.cytokines such as IL-4, IL5, IL9 and IL-13, bioactive peptides, cellsurface molecules, e.g. receptors, transporters, ion-channels, viral andbacterial proteins.

The term “monospecific” as used in the present invention means that thepolyvalent antibody of the present invention recognizes only oneantigen, all the antigen binding sites being identical.

The tem “bispecific” as used in the present invention means that thepolyvalent antibody of the present invention recognizes two differentepitopes on the same or on two different antigens.

The term “multispecific” as used in the present invention means that thepolyvalent antibody of the present invention recognizes multipledifferent epitopes on the same or on multiple different antigens.

The term “linker” as used in the present invention refers to a peptideadapted to connect the variable domains of the antibody constructs ofthe present invention. The peptide linker may contain any amino acids,the amino acids glycine (G) and serine (S) being preferred. The linkersmay be equal or differ from each other between and within the heavychain polypeptide and the light chain polypeptide. Furthermore, thelinker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 amino acids. A preferred peptide linkerunit for the heavy chain domains as for the light chain domains isGGGGS. The numbers of linker units of the heavy chain and of the lightchain may be equal (symmetrical order) or differ from each other(asymmetrical order).

A peptide linker is preferably long enough to provide an adequate degreeof flexibility to prevent the antibody moieties from interfering witheach others activity, for example by steric hindrance, to allow forproper protein folding and, if necessary, to allow the antibodymolecules to interact with two or more, possibly widely spaced,receptors on the same cell; yet it is preferably short enough to allowthe antibody moieties to remain stable in the cell.

Therefore, the length, composition and/or conformation of the peptidelinkers can readily be selected by one skilled in the art in order tooptimize the desired properties of the polyvalent antibody.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such asF_(v), F_(ab), F_(ab′), F_((ab′)2) or other target-binding subsequencesof antibodies) which contain sequences derived from non-humanimmunoglobulin, as compared to a human antibody. In general, thehumanized antibody will comprise substantially all of one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulintemplate sequence. The humanized antibody may also comprise at least aportion of an immunoglobulin constant region (F_(c)), typically that ofthe human immunoglobulin template chosen. In general, the goal is tohave an antibody molecule that is minimally immunogenic in a human.Thus, it is possible that one or more amino acids in one or more CDRsalso can be changed to one that is less immunogenic to a human host,without substantially minimizing the specific binding function of theone or more CDRs to IL-4 and/or IL-13. Alternatively, the FR can benon-human but those amino acids most immunogenic are replaced with onesless immunogenic. Nevertheless, CDR grafting, as discussed above, is notthe only way to obtain a humanized antibody. For example, modifying justthe CDR regions may be insufficient as it is not uncommon for frameworkresidues to have a role in determining the three-dimensional structureof the CDR loops and the overall affinity of the antibody for itsligand. Hence, any means can be practiced so that the non-human parentantibody molecule is modified to be one that is less immunogenic to ahuman, and global sequence identity with a human antibody is not alwaysa necessity. So, humanization also can be achieved, for example, by themere substitution of just a few residues, particularly those which areexposed on the antibody molecule and not buried within the molecule, andhence, not readily accessible to the host immune system. Such a methodis taught herein with respect to substituting “mobile” or “flexible”residues on the antibody molecule, the goal being to reduce or dampenthe immunogenicity of the resultant molecule without comprising thespecificity of the antibody for its epitope or determinant. See, forexample, Studnicka et al., Prot Eng 7(6)805-814, 1994; Mol 1 mm44:1986-1988, 2007; Sims et al., J Immunol 151:2296 (1993); Chothia etal., J Mol Biol 196:901 (1987); Carter et al., Proc Nail Acad Sci USA89:4285 (1992); Presta et al., J Immunol 151:2623 (1993), WO 2006/042333and U.S. Pat. No. 5,869,619.

A humanization method of interest is based on the impact of themolecular flexibility of the antibody during and at immune recognition.Protein flexibility is related to the molecular motion of the proteinmolecule. Protein flexibility is the ability of a whole protein, a partof a protein or a single amino acid residue to adopt an ensemble ofconformations which differ significantly from each other. Informationabout protein flexibility can be obtained by performing protein X-raycrystallography experiments (see, for example, Kundu et al. 2002,Biophys J 83:723-732.), nuclear magnetic resonance experiments (see, forexample, Freedberg et al., J Am Chem Soc 1998, 120(30:7916-7923) or byrunning molecular dynamics (MD) simulations. An MD simulation of aprotein is done on a computer and allows one to determine the motion ofall protein atoms over a period of time by calculating the physicalinteractions of the atoms with each other. The output of a MD simulationis the trajectory of the studied protein over the period of time of thesimulation. The trajectory is an ensemble of protein conformations, alsocalled snapshots, which are periodically sampled over the period of thesimulation, e.g. every 1 picosecond (ps). It is by analyzing theensemble of snapshots that one can quantify the flexibility of theprotein amino acid residues. Thus, a flexible residue is one whichadopts an ensemble of different conformations in the context of thepolypeptide within which that residue resides. MD methods are known inthe art, see, e.g., Brooks et al. “Proteins: A Theoretical Perspectiveof Dynamics, Structure and Thermodynamics” (Wiley, New York, 1988).Several software enable MD simulations, such as Amber (see Case et al.(2005) J Comp Chem 26:1668-1688), Charmm (see Brooks et al. (1983) JComp Chem 4:187-217; and MacKerell et al. (1998) in “The Encyclopedia ofComputational Chemistry” vol. 1:271-177, Schleyer et al., eds.Chichester: John Wiley & Sons) or Impact (see Rizzo et al. J Am ChemSoc; 2000; 122(51):12898-12900.)

Most protein complexes share a relatively large and planar buriedsurface and it has been shown that flexibility of binding partnersprovides the origin for their plasticity, enabling them toconformationally adapt to each other (Structure (2000) 8, R137-R142). Assuch, examples of “induced fit” have been shown to play a dominant rolein protein-protein interfaces. In addition, there is a steadilyincreasing body of data showing that proteins actually bind ligands ofdiverse shapes sizes and composition (Protein Science (2002) 11:184-187)and that the conformational diversity appears to be an essentialcomponent of the ability to recognize different partners (Science (2003)299, 1362-1367). Flexible residues are involved in the binding ofprotein-protein partners (Structure (2006) 14, 683-693).

The flexible residues can adopt a variety of conformations that providean ensemble of interaction areas that are likely to be recognized bymemory B cells and to trigger an immunogenic response. Thus, antibodycan be humanized by modifying a number of residues from the framework sothat the ensemble of conformations and of recognition areas displayed bythe modified antibody resemble as much as possible those adopted by ahuman antibody.

That can be achieved by modifying a limited number of residues by: (1)building a homology model of the parent mAb and running an MDsimulation; (2) analyzing the flexible residues and identification ofthe most flexible residues of a non-human antibody molecule, as well asidentifying residues or motifs likely to be a source of heterogeneity orof degradation reaction; (3) identifying a human antibody which displaysthe most similar ensemble of recognition areas as the parent antibody;(4) determining the flexible residues to be mutated, residues or motifslikely to be a source of heterogeneity and degradation are also mutated;and (5) checking for the presence of known T cell or B cell epitopes.The flexible residues can be found using an MD calculation as taughtherein using an implicit solvent model, which accounts for theinteraction of the water solvent with the protein atoms over the periodof time of the simulation. Once the set of flexible residues has beenidentified within the variable light and heavy chains, a set of humanheavy and light chain variable region frameworks that closely resemblethat of the antibody of interest are identified. That can be done, forexample, using a blast search on the set of flexible residues against adatabase of antibody human germline sequence. It can also be done bycomparing the dynamics of the parent mAb with the dynamics of a libraryof germline canonical structures. The CDR residues and neighboringresidues are excluded from the search to ensure high affinity for theantigen is preserved.

Flexible residues then are replaced. When several human residues showsimilar homologies, the selection is driven also by the nature of theresidues that are likely to affect the solution behavior of thehumanized antibody. For instance, polar residues will be preferred inexposed flexible loops over hydrophobic residues. Residues which are apotential source of instability and heterogeneity are also mutated evenif there are found in the CDRs. That will include exposed methionines assulfoxide formation can result from oxygen radicals, proteolyticcleavage of acid labile bonds such as those of the Asp-Pro dipeptide(Drug Dev Res (2004) 61:137-154), deamidation sites found with anexposed asparagine residue followed by a small amino acid, such as Gly,Ser, Ala, His, Asn or Cys (J Chromatog (2006) 837:35-43) andN-glycosylation sites, such as the Asn-X-Ser/Thr site. Typically,exposed methionines will be substituted by a Leu, exposed asparagineswill be replaced by a glutamine or by an aspartate, or the subsequentresidue will be changed. For the glycosylation site (Asn-X-Ser/Thr),either the Asn or the Ser/Thr residue will be changed.

The resulting composite sequence is checked for the presence of known Bcell or linear T-cell epitopes. A search is performed, for example, withthe publicly available IEDB. If a known epitope is found within thecomposite sequence, another set of human sequences is retrieved andsubstituted

Unlike the resurfacing method of U.S. Pat. No. 5,639,641, bothB-cell-mediated and T-cell-mediated immunogenic responses are addressedby the method. The method also avoids the issue of loss of activity thatis sometimes observed with CDR grafting (U.S. Pat. No. 5,530,101). Inaddition, stability and solubility issues also are considered in theengineering and selection process, resulting in an antibody that isoptimized for low immunogenicity, high antigen affinity and improvedbiophysical properties.

Strategies and methods for resurfacing antibodies, and other methods forreducing immunogenicity of antibodies within a different host, aredisclosed, for example, in U.S. Pat. No. 5,639,641. Briefly, in apreferred method, (1) position alignments of a pool of antibody heavyand light chain variable regions are generated to yield heavy and lightchain variable region framework surface exposed positions, wherein thealignment positions for all variable regions are at least about 98%identical; (2) a set of heavy and light chain variable region frameworksurface exposed amino acid residues is defined for a non-human, such asa rodent antibody (or fragment thereof); (3) a set of heavy and lightchain variable region framework surface exposed amino acid residues thatis most closely identical to the set of rodent surface exposed aminoacid residues is identified; and (4) the set of heavy and light chainvariable region framework surface exposed amino acid residues defined instep (2) is substituted with the set of heavy and light chain variableregion framework surface exposed amino acid residues identified in step(3), except for those amino acid residues that are within 5 Å of anyatom of any residue of a CDR of the rodent antibody, to yield ahumanized, such as a rodent antibody retaining binding specificity.

Antibodies can be humanized by a variety of other techniques includingCDR grafting (EPO 0 239 400; WO 91/09967; and U.S. Pat. Nos. 5,530,101and 5,585,089), veneering or resurfacing (EPO 0 592 106; EPO 0 519 596;Padlan, 1991, Molec 1 mm 28(4/5):489-498; Studnicka et al., 1994, ProtEng 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973) and chainshuffling (U.S. Pat. No. 5,565,332). Human antibodies can be made by avariety of methods known in the art including, but not limited to, phagedisplay methods, see U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806 and5,814,318; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735 and WO 91/10741, using transgenic animals, such asrodents, using chimeric cells and so on.

“Antibody homolog” or “homolog” refers to any molecule whichspecifically binds IL-4 and/or IL-13 as taught herein. Thus, an antibodyhomolog includes native or recombinant antibody, whether modified ornot, portions of antibodies that retain the biological properties ofinterest, such as binding IL-4 or IL-13, such as an F_(ab) or F_(v)molecule, a single chain antibody, a polypeptide carrying one or moreCDR regions and so on. The amino acid sequence of the homolog need notbe identical to that of the naturally occurring antibody but can bealtered or modified to carry substitute amino acids, inserted aminoacids, deleted amino acids, amino acids other than the twenty normallyfound in proteins and so on to obtain a polypeptide with enhanced orother beneficial properties.

Antibodies with homologous sequences are those antibodies with aminoacid sequences that have sequence homology with the amino acid sequenceof a IL-4, IL-13 or bispecific IL-4/IL-13 antibody of the presentinvention. Preferably, homology is with the amino acid sequence of thevariable regions of an antibody of the present invention. “Sequencehomology” as applied to an amino acid sequence herein is defined as asequence with at least about 90%, 91%, 92%, 93%, 94% or more sequencehomology, and more preferably at least about 95%, 96%, 97%, 98% or 99%sequence homology to another amino acid sequence, as determined, forexample, by the FASTA search method in accordance with Pearson & Lipman,Proc Natl Acad Sci USA 85, 2444-2448 (1988).

A chimeric antibody is one with different portions of an antibodyderived from different sources, such as different antibodies, differentclasses of antibody, different animal species, for example, an antibodyhaving a variable region derived from a murine monoclonal antibodypaired with a human immunoglobulin constant region and so on. Thus, ahumanized antibody is a species of chimeric antibody. Methods forproducing chimeric antibodies are known in the art, see, e.g., Morrison,1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies etal., 1989, J Immunol Methods 125:191-202; and U.S. Pat. Nos. 5,807,715,4,816,567, and 4,816,397.

Artificial antibodies include scFv fragments, chimeric antibodies,diabodies, triabodies, tetrabodies and mru (see reviews by Winter &Milstein, 1991, Nature 349:293-299; and Hudson, 1999, Curr Opin 1 mm11:548-557), each with antigen-binding or epitope-binding ability. Inthe single chain F_(v) fragment (scF_(v)), the V_(H) and V_(L) domainsof an antibody are linked by a flexible peptide. Typically, the linkeris a peptide of about 15 amino acids. If the linker is much smaller, forexample, 5 amino acids, diabodies are formed. The smallest binding unitof an antibody is a CDR, typically the CDR2 of the heavy chain which hassufficient specific recognition and binding capacity. Such a fragment iscalled a molecular recognition unit or mru. Several such mrus can belinked together with short linker peptides, therefore forming anartificial binding protein with higher avidity than a single mru.

Also included within the scope of the invention are functionalequivalents of an antibody of interest. The term “functionalequivalents” includes antibodies with homologous sequences, antibodyhomologs, chimeric antibodies, artificial antibodies and modifiedantibodies, for example, wherein each functional equivalent is definedby the ability to bind to IL-4 and/or IL-13, inhibiting IL-4 and/orIL-13 signaling ability or function, or inhibiting binding of IL-4and/or IL-13 to its receptor. The skilled artisan will understand thatthere is an overlap in the group of molecules termed “antibodyfragments” and the group termed “functional equivalents.” Methods ofproducing functional equivalents which retain IL-4 and/or IL-13 bindingability are known to the person skilled in the art and are disclosed,for example, in WO 93/21319, EPO Ser. No. 239,400, WO 89/09622, EPO Ser.No. 338,745 and EPO Ser. No. 332,424.

The functional equivalents of the present application also includemodified antibodies, e.g., antibodies modified by the covalentattachment of any type of molecule to the antibody. For example,modified antibodies include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, deamidation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand, linkage to a toxinor cytotoxic moiety or other protein etc. The covalent attachment neednot yield an antibody that is immune from generating an anti-idiotypicresponse. The modifications may be achieved by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis etc. Additionally, the modifiedantibodies may contain one or more non-classical amino acids.

Many techniques are available to one of ordinary skill in the art whichpermit the optimization of binding affinity. Typically, the techniquesinvolve substitution of various amino acid residues at the site ofinterest, followed by a screening analysis of binding affinity of themutant polypeptide for the cognate antigen or epitope.

Once the antibody is identified and isolated, it is often useful togenerate a variant antibody or mutant, or mutein, wherein one or moreamino acid residues are altered, for example, in one or more of thehypervariable regions of the antibody. Alternatively, or in addition,one or more alterations (e.g., substitutions) of framework residues maybe introduced in the antibody where these result in an improvement inthe binding affinity of the antibody mutant for IL-4 and/or IL-13.Examples of framework region residues that can be modified include thosewhich non-covalently bind antigen directly (Amit et al., Science233:747-753 (1986)); interact with/affect the conformation of a CDR(Chothia et al., J Mol Biol 196:901-917 (1987)); and/or participate inthe V_(L)-V_(H) interface (EP 239 400). In certain embodiments,modification of one or more of such framework region residues results inan enhancement of the binding affinity of the antibody for the cognateantigen. For example, from about one to about five framework residuesmay be altered in this embodiment of the invention. Sometimes, this maybe sufficient to yield an antibody mutant suitable for use inpreclinical trials, even where none of the hypervariable region residueshave been altered. Normally, however, the antibody mutant can compriseone or more hypervariable region alteration(s). The constant regionsalso can be altered to obtain desirable or more desirable effectorproperties.

The hypervariable region residues which are altered may be changedrandomly, especially where the starting binding affinity of the parentantibody is such that randomly-produced antibody mutants can be readilyscreened for altered binding in an assay as taught herein.

One procedure for obtaining antibody mutants, such as CDR mutants, is“alanine scanning mutagenesis” (Cunningham & Wells, Science244:1081-1085 (1989); and Cunningham & Wells, Proc Nat Acad Sci USA84:6434-6437 (1991)). One or more of the hypervariable region residue(s)are replaced by alanine or polyalanine residue(s). Those hypervariableregion residue(s) demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other mutationsat or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. Similarsubstitutions can be attempted with other amino acids, depending on thedesired property of the scanned residues.

A more systematic method for identifying amino acid residues to modifycomprises identifying hypervariable region residues involved in bindingIL-4 and/or IL-13 and those hypervariable region residues with little orno involvement with IL-4 and/or IL-13 binding. An alanine scan of thenon-binding hypervariable region residues is performed, with each alamutant tested for enhancing binding to IL-4 and/or IL-13. In anotherembodiment, those residue(s) significantly involved in binding IL-4and/or IL-13 are selected to be modified. Modification can involvedeletion of a residue or insertion of one or more residues adjacent to aresidue of interest. However, normally the modification involvessubstitution of the residue by another amino acid. A conservativesubstitution can be a first substitution. If such a substitution resultsin a change in biological activity (e.g., binding affinity), thenanother conservative substitution can be made to determine if moresubstantial changes are obtained.

Even more substantial modification in an antibody range and presentationof biological properties can be accomplished by selecting an amino acidthat differs more substantially in properties from that normallyresident at a site. Thus, such a substitution can be made whilemaintaining: (a) the structure of the polypeptide backbone in the areaof the substitution, for example, as a sheet or helical conformation;(b) the charge or hydrophobicity of the molecule at the target site, or(c) the bulk of the side chain.

For example, the naturally occurring amino acids can be divided intogroups based on common side chain properties:

(1) hydrophobic: methionine (M or met), alanine (A or ala), valine (V orval), leucine (L or leu) and isoleucine (I or ile);

(2) neutral, hydrophilic: cysteine (C or cys), serine (S or ser),threonine (T or thr), asparagine (N or asn) and glutamine (Q or gln);

(3) acidic: aspartic acid (D or asp) and glutamic acid (E or glu);

(4) basic: histidine (H or his), lysine (K or lys) and arginine (R orarg);

(5) residues that influence chain orientation: glycine (G or gly) andproline (P or pro), and

(6) aromatic: tryptophan (W or trp), tyrosine (Y or tyr) andphenylalanine (F or phe).

Non-conservative substitutions can entail exchanging an amino acid withan amino acid from another group. Conservative substitutions can entailexchange of one amino acid for another within a group.

Preferred amino acid substitutions include those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity and (4) confer or modify otherphysico-chemical or functional properties of such analogs. Analogs caninclude various muteins of a sequence other than the naturally occurringpeptide sequence. For example, single or multiple amino acidsubstitutions (preferably conservative amino acid substitutions) may bemade in the naturally-occurring sequence (preferably in the portion ofthe polypeptide outside the domain (s) forming intermolecular contacts.A conservative amino acid substitution should not substantially changethe structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence) unless of a change in the bulk orconformation of the R group or side chain, Proteins, Structures andMolecular Principles (Creighton, ed., W. H. Freeman and Company, NewYork (1984)); Introduction to Protein Structure (Branden & Tooze, eds.,Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature354:105 (1991).

Ordinarily, the antibody mutant with improved biological properties willhave an amino acid sequence having at least 75% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the parent anti-human IL-4 and/orIL-13 antibody, at least 80%, at least 85%, at least 90% and often atleast 95% identity. Identity or similarity with respect to parentantibody sequence is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical (i.e., sameresidue) or similar (i.e., amino acid residue from the same group basedon common side-chain properties, supra) with the parent antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity.

Alternatively, antibody mutants can be generated by systematic mutationof the FR and CDR regions of the heavy and light chains, or the F_(c)region of the anti-IL-4, anti-IL-13 or bispecific IL-4/IL-13 antibody.Another procedure for generating antibody mutants involves the use ofaffinity maturation using phage display (Hawkins et al., J Mol Biol254:889-896 (1992) and Lowman et al., Biochemistry 30(45):10832-10838(1991)). Bacteriophage coat-protein fusions (Smith, Science 228:1315(1985); Scott & Smith, Science 249:386 (1990); Cwirla et al. Proc NatlAcad Sci USA 8:309 (1990); Devlin et al. Science 249:404 (1990); Wells &Lowman, Curr Opin Struct Biol 2:597 (1992); and U.S. Pat. No. 5,223,409)are known to be useful for linking the phenotype of displayed proteinsor peptides to the genotype of bacteriophage particles which encodethem. The F_(ab) domains of antibodies have also been displayed on phage(McCafferty et al., Nature 348: 552 (1990); Barbas et al. Proc Natl AcadSci USA 88:7978 (1991); and Garrard et al. Biotechnol 9:1373 (1991)).

Monovalent phage display consists of displaying a set of proteinvariants as fusions of a bacteriophage coat protein on phage particles(Bass et al., Proteins 8:309 (1990). Affinity maturation, or improvementof equilibrium binding affinities of various proteins, has previouslybeen achieved through successive application of mutagenesis, monovalentphage display and functional analysis (Lowman & Wells, J Mol Biol234:564 578 (1993); and U.S. Pat. No. 5,534,617), for example, byfocusing on the CDR regions of antibodies (Barbas et al., Proc Natl AcadSci USA 91:3809 (1994); and Yang et al., J Mol Biol 254:392 (1995)).

Libraries of many (for example, 10⁶ or more) protein variants, differingat defined positions in the sequence, can be constructed onbacteriophage particles, each of which contains DNA encoding theparticular protein variant. After cycles of affinity purification, usingan immobilized antigen, individual bacteriophage clones are isolated,and the amino acid sequence of the displayed protein is deduced from theDNA.

Following production of the antibody mutant, the biological activity ofthat molecule relative to the parent antibody can be determined astaught herein. As noted above, that may involve determining the bindingaffinity and/or other biological activities or physical properties ofthe antibody. In a preferred embodiment of the invention, a panel ofantibody mutants is prepared and screened for binding affinity for theantigen. One or more of the antibody mutants selected from the screenare optionally subjected to one or more further biological activityassays to confirm that the antibody mutant(s) have new or improvedproperties. In preferred embodiments, the antibody mutant retains theability to bind IL-4 and/or IL-13 with a binding affinity similar to orbetter/higher than that of the parent antibody.

The antibody mutant(s) so selected may be subjected to furthermodifications, often depending on the intended use of the antibody. Suchmodifications may involve further alteration of the amino acid sequence,fusion to heterologous polypeptide(s) and/or covalent modifications. Forexample, a cysteine residue not involved in maintaining the properconformation of the antibody mutant may be substituted, generally withserine, to improve the oxidative stability of the molecule and toprevent aberrant cross-linking. Conversely, a cysteine may be added tothe antibody to improve stability (particularly where the antibody is anantibody fragment such as an F_(v) fragment).

Another type of antibody mutant has an altered glycosylation pattern.That may be achieved by deleting one or more carbohydrate moieties foundin the antibody and/or by adding one or more glycosylation sites thatare not present in the antibody. Glycosylation of antibodies istypically either N-linked to Asn or O-linked to Ser or Thr. Thetripeptide sequences, asparagine-X-serine and asparagine-X-thrconine,where X is any amino acid except proline, are common recognitionsequences for enzymatic attachment of a carbohydrate moiety to theasparagine side chain. N-acetylgalactosamine, galactose, fucose orxylose, for example, are bonded to a hydroxyamino acid, most commonlyserine or threonine, although 5-hydroxyproline or 5-hydroxylysine alsomay be used. Addition or substitution of one or more serine or threonineresidues to the sequence of the original antibody can enhance thelikelihood of O-linked glycosylation.

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of theantibody. For example, cysteine residue(s) may be introduced in theF_(c) region, thereby allowing interchain disulfide bond formation inthat region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC), see Caronet al., J Exp Med 176:1191-1195 (1992) and Shopes, Immunol 148:2918-2922(1993). Alternatively, an antibody can be engineered which has dualF_(c) regions and may thereby have enhanced complement lysis and ADCCcapabilities, see Stevenson et al., Anti-Cancer Drug Design 3: 219 230(1989).

Covalent modifications of the antibody are included within the scope ofthe invention. Such may be made by chemical synthesis or by enzymatic orchemical cleavage of the antibody, if applicable. Other types ofcovalent modifications of the antibody are introduced into the moleculeby reacting targeted amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor with the N-terminal or C-terminal residue.

Cysteinyl residues can be reacted with α-haloacetates (and correspondingamines), such as chloroacetic acid or chloroacetamide, to yieldcarboxylmethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso can be derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl) propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercura-4-nitrophenol orchloro-7-nitrobenzo-2-oxa-1,3-diazole, for example.

Histidyl residues can be derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0. p-bromophenacyl bromide also can beused, the reaction is preferably performed in 0.1 M sodium cacodylate atpH 6.0.

Lysinyl and a terminal residues can be reacted with succinic or othercarboxylic acid anhydrides to reverse the charge of the residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters, such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea and 2,4-pentanedione, and the amino acid can betransaminase-catalyzed with glyoxylate.

Arginyl residues can be modified by reaction with one or severalconventional reagents, such as phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residuesoften requires alkaline reaction conditions. Furthermore, the reagentsmay react with lysine as well as the arginine ε-amino group.

The specific modification of tyrosyl residues can be made with aromaticdiazonium compounds or tetranitromethane. For example, N-acetylimidizoleand tetranitromethane are used to form O-acetyl tyrosyl species and3-nitro derivatives, respectively. Tyrosyl residues can be iodinatedusing ¹²⁵I or ¹³¹I to prepare labeled proteins for use in aradioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) can be modified by reactionwith carbodiimides (R—N═C═C—R′), where R and R′ can be different alkylgroups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively, underneutral or basic conditions. The deamidated form of those residues fallswithin the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of serinyl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (Creighton, Proteins: Structure and Molecular Properties,W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of theN-terminal amine and amidation of any C-terminal carboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. Those procedures donot require production of the antibody in a host cell that hasglycosylation capabilities for N-linked or O-linked glycosylation.Depending on the coupling mode used, the sugar(s) may be attached to:(a) arginine and histidine; (b) free carboxyl groups; (c) freesulthydryl groups, such as those of cysteine; (d) free hydroxyl groups,such as those of scrinc, thrconinc or hydroxyprolinc; (c) aromaticresidues such as those of phenylalanine, tyrosine or tryptophan; or (f)the amide group of glutamine. Such methods are described in WO 87/05330and in Aplin & Wriston, CRC Crit Rev Biochem, pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylation, forexample, can require exposure of the antibody to the compound,trifluoromethanesulfonic acid, or an equivalent compound, resulting incleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described, for example, inHakimuddin et al. Arch Biochem Biophys 259:52 (1987) and in Edge et al.,Anal Biochem 118:131 (1981). Enzymatic cleavage of carbohydrate moietieson antibodies can be achieved by any of a variety of endoglycosidasesand exoglycosidases as described, for example, in Thotakura et al., MethEnzymol 138:350 (1987).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol or polyoxylalkylenes, in themanner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

Another technique preferred for obtaining mutants or muteins is affinitymaturation by phage display (Hawkins et al., J Mol Biol 254:889-896(1992); and Lowman et al., Biochemistry 30(45):10832-10838 (1991)).Briefly, several hypervariable region sites (e.g., 6-7 sites) aremutated to generate all possible amino acid substitutions at each site.The antibody mutants thus generated are displayed in monovalent fashionon phage particles as fusions to a protein found on the particles. Thephage expressing the various mutants can be cycled through rounds ofbinding selection, followed by isolation and sequencing of those mutantswhich display high affinity.

The method of selecting novel binding polypeptides can utilize a libraryof structurally related polypeptides. The library of structurallyrelated polypeptides, for example, fused to a phage coat protein, isproduced by mutagenesis, and is displayed on the surface of theparticle. The particles then are contacted with a target molecule andthose particles having the highest affinity for the target are separatedfrom those of lower affinity. The high affinity binders then areamplified by infection of a suitable bacterial host and the competitivebinding step is repeated. The process is repeated until polypeptides ofthe desired affinity are obtained.

Alternatively, multivalent phage (McCafferty et al. (1990) Nature348:552-554; and Clackson et al. (1991) Nature 352:624-628) also can beused to express random point mutations (for example, generated by use ofan error-prone DNA polymerase) to generate a library of phage antibodyfragments which then could be screened for affinity to IL-4 and/orIL-13, Hawkins et al., (1992) J Mol Biol 254:889-896.

Preferably, during the affinity maturation process, the replicableexpression vector is under tight control of a transcription regulatoryelement, and the culturing conditions are adjusted so the amount ornumber of particles displaying more than one copy of the fusion proteinis less than about 1%. Also preferably, the amount of particlesdisplaying more than one copy of the fusion protein is less than 10% ofthe amount of particles displaying a single copy of the fusion protein.Preferably the amount is less than 20%.

Functional equivalents may be produced by interchanging different CDRsof different antibody chains within a framework or a composite FRderived from plural antibodies. Thus, for example, different classes ofantibody are possible for a given set of CDRs by substitution ofdifferent heavy chains, for example, IgG₁₋₄, IgM, IgA₁₋₂ or IgD, toyield differing IL-4 and/or IL-13 antibody types and isotypes.Similarly, artificial antibodies within the scope of the invention maybe produced by embedding a given set of CDRs within an entirelysynthetic framework.

The antibody fragments and functional equivalents of the presentinvention encompass those molecules with a detectable degree of specificbinding to IL-4 and/or IL-13. A detectable degree of binding includesall values in the range of at least 10-100%, preferably at least 50%,60% or 70%, more preferably at least 75%, 80%, 85%, 90%, 95% or 99% ofthe binding ability of an antibody of interest. Also included areequivalents with an affinity greater than 100% that of an antibody ofinterest.

The CDRs generally are of importance for epitope recognition andantibody binding. However, changes may be made to residues that comprisethe CDRs without interfering with the ability of the antibody torecognize and to bind the cognate epitope. For example, changes that donot impact epitope recognition, yet increase the binding affinity of theantibody for the epitope, may be made. Several studies have surveyed theeffects of introducing one or more amino acid changes at variouspositions in the sequence of an antibody, based on the knowledge of theprimary antibody sequence, on the properties thereof, such as bindingand level of expression (Yang et al., 1995, J Mol Biol 254:392-403;Rader et al., 1998, Proc Natl Acad Sci USA 95:8910-8915; and Vaughan etal., 1998, Nature Biotechnology 16, 535-539).

Thus, equivalents of an antibody of interest can be generated bychanging the sequences of the heavy and light chain genes in the CDR1,CDR2 or CDR3, or framework regions, using methods such asoligonucleotide-mediated site-directed mutagenesis, cassettemutagenesis, error-prone PCR, DNA shuffling or mutator-strains of E.coli (Vaughan et al., 1998, Nat Biotech 16:535-539; and Adey et al.,1996, Chap. 16, pp. 277-291, in Phage Display of Peptides and Proteins,eds. Kay et al., Academic Press). The methods of changing the nucleicacid sequence of the primary antibody can result in antibodies withimproved affinity (Gram et al., 1992, Proc Natl Acad Sci USA89:3576-3580; Boder et al., 2000, Proc Natl Acad Sci USA 97:10701-10705;Davies & Riechmann, 1996, Immunotech 2:169-179; Thompson et al., 1996, JMol Biol 256:77-88; Short et al., 2002, J Biol Chem 277:16365-16370; andFurukawa et al., 2001, J Biol Chem 276:27622-27628).

Repeated cycles of “polypeptide selection” can be used to select forhigher and higher affinity binding by, for example, the selection ofmultiple amino acid changes which are selected by multiple selections ofcycles. Following a first round of selection, involving a first regionof selection of amino acids in the ligand or antibody polypeptide,additional rounds of selection in other regions or amino acids of theligand are conducted. The cycles of selection are repeated until thedesired affinity properties are achieved.

Improved antibodies also include those antibodies having improvedcharacteristics that are prepared by the standard techniques of animalimmunization, hybridoma formation and selection for antibodies withspecific characteristics.

“Antagonist” refers to a molecule capable of inhibiting one or morebiological activities of a target molecule, such as signaling by IL-4and/or IL-13. Antagonists may interfere with the binding of a receptorto a ligand and vice versa, by incapacitating or killing cells activatedby a ligand, and/or by interfering with receptor or ligand activation(e.g., tyrosine kinase activation) or signal transduction after ligandbinding to a receptor. The antagonist may completely blockreceptor-ligand interactions or may substantially reduce suchinteractions.

“Agonist” refers to a compound, including a protein, a polypeptide, apeptide, an antibody, an antibody fragment, a conjugate, a largemolecule, a small molecule, which activates one or more biologicalactivities of IL-4 and/or IL-13. Agonists may interact with the bindingof a receptor to a ligand and vice versa, by acting as a mitogen ofcells activated by a ligand, and/or by interfering with cellinactivation or signal transduction inhibition after ligand binding to areceptor. All such points of intervention by an agonist shall beconsidered equivalent for purposes of the instant invention.

The terms “cell,” “cell line,” and “cell culture” include progenythereof. It is also understood that all progeny may not be preciselyidentical, such as in DNA content, due to deliberate or inadvertentmutation. Variant progeny that have the same function or biologicalproperty of interest, as screened for in the original cell, areincluded.

The term “vector” means a nucleic acid construct, a carrier, containinga nucleic acid, the transgene, the foreign gene or the gene of interest,which can be operably linked to suitable control sequences forexpression of the transgene in a suitable host. Such control sequencesinclude, for example, a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable mRNA ribosome binding sites and sequences which control thetermination of transcription and translation. The vector may be aplasmid, a phage particle or just a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may in some instances, integrateinto the host cell genome. In the present specification, “plasmid” and“vector” are used interchangeably, as the plasmid is a commonly usedform of vector. However, the invention is intended to include such otherforms of vectors which serve equivalent carrier function as and whichare, or become, known in the art, such as viruses, synthetics moleculesthat carry nucleic acids, liposomes and the like.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including human, domestic and farm animals, nonhuman primates,and zoo, sports or pet animals, such as dogs, horses, cats, cows etc.

The antibodies of interest can be screened or can be used in an assay asdescribed herein or as known in the art. Often, such assays require areagent to be detectable, that is, for example, labeled. The word“label” when used herein refers to a detectable compound or compositionwhich can be conjugated directly or indirectly to a molecule or protein,e.g., an antibody. The label may itself be detectable (e.g.,radioisotope labels, particles or fluorescent labels) or, in the case ofan enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

As used herein, “solid phase” means a non-aqueous matrix to which anentity or molecule, such as the antibody of the instant invention, canadhere. Example of solid phases encompassed herein include those formedpartially or entirely of glass (e.g., controlled pore glass),polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinylalcohol and silicones. In certain embodiments, depending on the context,the solid phase can comprise the well of an assay plate; in others canbe used in a purification column (e.g., an affinity chromatographycolumn). Thus, the solid phase can be a paper, a bead, a plastic, a chipand so on, can be made from a variety of materials, such asnitrocellulose, agarose, polystyrene, polypropylene, silicon and so on,and can be in a variety of configurations.

The gene or a cDNA encoding IL-4 and IL-13 are known in the art, may becloned in a plasmid or other expression vector and expressed in any of anumber of expression systems according to methods well known to those ofskill in the art, and see below, for example.

Nucleic acid molecules encoding amino acid sequence mutants can beprepared by a variety of methods known in the art. The methods include,but are not limited to, oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis and cassette mutagenesis of an earlierprepared mutant or a non-mutant version of the molecule of interest,(see, for example, Kunkel, Proc Natl Acad Sci USA 82:488 (1985)).

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention, a single chain antibody of the invention oran antibody mutein of the invention) includes construction of anexpression vector containing a polynucleotide that encodes the antibodyor a fragment of the antibody as described herein. Once a polynucleotideencoding an antibody molecule has been obtained, the vector for theproduction of the antibody may be produced by recombinant DNA technologyas known in the art. An expression vector is constructed containingantibody coding sequences and appropriate transcriptional andtranslational control signals. The methods include, for example, invitro recombinant DNA techniques, synthetic techniques and in vivogenetic recombination.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells then are cultured by conventionaltechniques to produce an antibody or fragment of the invention. In oneaspect of the invention, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed herein.

A variety of host/expression vector systems may be utilized to expressthe antibody molecules of the invention. Such expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ.Bacterial cells, such as E. coli, and eukaryotic cells are commonly usedfor the expression of a recombinant antibody molecule, especially forthe expression of whole recombinant antibody molecule. For example,mammal cells such as CHO cells, in conjunction with a vector, such asone carrying the major intermediate early gene promoter element fromhuman cytomegalovirus, are an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); and Cockett et al., Bio/Technology8:2 (1990)). Plants and plant cell culture, insect cells and so on alsocan be used to make the proteins of interest, as known in the art.

In addition, a host cell is chosen which modulates the expression of theinserted sequences, or modifies and processes the gene product in thespecific fashion desired. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of protein products may be important for thefunction of the protein. Different host cells have characteristic andspecific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the expressed antibody of interest. Hence, eukaryotic hostcells which possess the cellular machinery for proper processing of theprimary transcript, glycosylation and phosphorylation of the geneproduct may be used. Such mammalian host cells include, but are notlimited to, CHO, COS, 293, 3T3 or myeloma cells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites etc.) and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for one to two days in an enriched media, and then aremoved to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into a chromosome and be expanded into a cellline. Such engineered cell lines not only are useful for antibodyproduction but are useful in screening and evaluation of compounds thatinteract directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe Herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska etal., Proc Natl Acad Sci USA 48:202 (1992)), glutamate synthase selectionin the presence of methionine sulfoximide (Adv Drug Del Rev 58, 671,2006 and see the website or literature of Lonza Group Ltd.) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes in tk,hgprt or aprt cells, respectively. Also, antimetabolite resistance canbe used as the basis of selection for the following genes: dhfr, whichconfers resistance to methotrexate (Wigler et al., Proc Natl Acad SciUSA 77:357 (1980); O'Hare et al., Proc Natl Acad Sci USA 78:1527(1981)); gpt, which confers resistance to mycophenolic acid (Mulligan etal., Proc Natl Acad Sci USA 78:2072 (1981)); neo, which confersresistance to the aminoglycoside, G-418 (Wu et al., Biotherapy 3:87(1991)); and hygro, which confers resistance to hygromycin (Santerre etal., Gene 30:147 (1984)). Methods known in the art of recombinant DNAtechnology may be routinely applied to select the desired recombinantclone, and such methods are described, for example, in Ausubel et al.,eds., Current Protocols in Molecular Biology, John Wiley & Sons (1993);Kriegler, Gene Transfer and Expression, A Laboratory Manual, StocktonPress (1990); Dracopoli et al., eds., Current Protocols in HumanGenetics, John Wiley & Sons (1994); and Colberre-Garapin et al., J MolBiol 150:1 (1981).

The expression levels of an antibody molecule can be increased by vectoramplification (for example, see Bebbington et al., in DNA Cloning, Vol.3. Academic Press (1987)). When a marker in the vector system expressingantibody is amplifiable, an increase in the level of inhibitor presentin the culture will increase the number of copies of the marker gene.Since the amplified region is associated with the antibody gene,production of the antibody will also increase (Crouse et al., Mol CellBiol 3:257 (1983)).

The host cell may be co-transfected with two or more expression vectorsof the invention, for example, the first vector encoding a heavychain-derived polypeptide and the second vector encoding a lightchain-derived polypeptide. The two vectors may contain identicalselectable markers which enable equal expression of heavy and lightchain polypeptides. Alternatively, a single vector may be used whichencodes, and is capable of expressing, both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, Nature 322:52 (1986); and Kohler, Proc Natl Acad Sci USA77:2197 (1980)). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for IL-4 and/or IL-13 afterProtein A and size-exclusion chromatography and so on), centrifugation,differential solubility or by any other standard technique for thepurification of proteins. In addition, the antibodies of the instantinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The antibodies of the present invention may be generated by any suitablemethod known in the art. The antibodies of the present invention maycomprise polyclonal antibodies, although because of the modification ofantibodies to optimize use in human, as well as to optimize the use ofthe antibody per se, monoclonal antibodies are preferred because of easeof production and manipulation of particular proteins. Methods ofpreparing polyclonal antibodies are known to the skilled artisan (Harlowet al., Antibodies: a Laboratory Manual, Cold Spring Harbor LaboratoryPress, 2nd ed. (1988)).

The antibodies of the present invention preferably comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomatechnology, such as described by Kohler et al., Nature 256:495 (1975);U.S. Pat. No. 4,376,110; Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 2nd ed. (1988) and Hammerling etal., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier (1981),recombinant DNA methods, for example, making and using transfectomas, orother methods known to the artisan. Other examples of methods which maybe employed for producing monoclonal antibodies include, but are notlimited to, the human B-cell hybridoma technique (Kosbor et al.,Immunology Today 4:72 (1983); and Cole et al., Proc Natl Acad Sci USA80:2026 (1983)), and the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss(1985)). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA and IgD, and any subclass thereof. The hybridomaproducing the mAb of the invention may be cultivated in vitro or invivo.

In the hybridoma model, a host such as a mouse, a humanized mouse, atransgenic mouse with human immune system genes, hamster, rabbit, rat,camel or any other appropriate host animal, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatspecifically bind to IL-4 or IL-13. Alternatively, lymphocytes may beimmunized in vitro. Lymphocytes then are fused with mycloma cells usinga suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, pp. 59-103 (1986)).

Generally, in making antibody-producing hybridomas, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine or human origin. Typically, a rat or mouse myeloma cell line isemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin and thymidine (“HATmedium”), substances that prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asthose derived from the MOPC-21 and MPC-11 mouse tumors available fromthe Salk Institute Cell Distribution Center, San Diego, Calif. andSP2/0, FO or X63-Ag8-653 cells available from the American Type CultureCollection, Manassas, Va.

Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, JImmunol 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc, pp. 51-63(1987)). The mouse myeloma cell line NSO may also be used (EuropeanCollection of Cell Cultures, Salisbury, Wilshire, UK).

Another alternative is to use electrical fusion rather than chemicalfusion to form hybridomas. Instead of fusion, a B cell can beimmortalized using, for example, Epstein Barr Virus or anothertransforming gene, see, e.g., Zurawaki et al., in Monoclonal Antibodies,ed., Kennett et al., Plenum Press, pp. 19-33. (1980). Transgenic miceexpressing immunoglobulins and severe combined immunodeficient (SCID)mice transplanted with human B lymphocytes also can be used.

The culture medium in which hybridoma cells are grown is assayed forproduction of monoclonal antibodies directed against IL-4 and/or IL-13.The binding specificity of monoclonal antibodies produced by hybridomacells may be determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA), fluorocytometric analysis (FACS)or enzyme-linked immunosorbent assay (ELISA). Such techniques are knownin the art and are within the skill of the artisan. The binding affinityof the monoclonal antibody to IL-4 and/or IL-13 can, for example, bedetermined by a Scatchard analysis (Munson et al., Anal Biochem 107:220(1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,pp. 59-103 (1986)). Suitable culture media include, for example,Dulbecco's Modified Eagle's Medium (D-MEM) or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated or isolated from the culture medium, ascites fluid or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, protein G-Sepharose, hydroxylapatitechromatography, gel exclusion chromatography, gel electrophoresis,dialysis or affinity chromatography.

A variety of methods exist in the art for the production of monoclonalantibodies and thus, the invention is not limited to their soleproduction in hybridomas. For example, the monoclonal antibodies may bemade by recombinant DNA methods, such as those described in U.S. Pat.No. 4,816,567. In this context, the term “monoclonal antibody” refers toan antibody derived from a single eukaryotic, phage or prokaryoticclone.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies, or such chainsfrom human, humanized or other sources) (Innis et al. in PCR Protocols.A Guide to Methods and Applications, Academic (1990), and Sanger et al.,Proc Natl Acad Sci 74:5463 (1977)). The hybridoma cells serve as asource of such DNA. Once isolated, the DNA may be placed into expressionvectors, which are then transfected into host cells such as E. colicells, NSO cells, COS cells, Chinese hamster ovary (CHO) cells ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. The DNA also may be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567; andMorrison et al., Proc Natl Acad Sci USA 81:6851 (1984)) or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneIL-4 or IL-13 combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the F_(c) region so as to prevent heavy chain cross-linkingAlternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent cross-linking

Antibody fragments which recognize specific epitopes may be generated byknown techniques. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,J Biochem Biophys Methods 24:107 (1992); and Brennan et al., Science229:81 (1985)). For example, F_(ab) and F_((ab′)2) fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce F_(ab) fragments) orpepsin (to produce F_((ab′)2) fragments). F_((ab′)2) fragments containthe variable region, the light chain constant region and the C_(H1)domain of the heavy chain. However, those fragments can be produceddirectly by recombinant host cells. For example, the antibody fragmentscan be isolated from an antibody phage library. Alternatively,F_((ab′)2)-SH fragments can be directly recovered from E. coli andchemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163 (1992). According to another approach, F_((ab′)2)fragments can be isolated directly from recombinant host cell culture.Other techniques for the production of antibody fragments will beapparent to the skilled practitioner. In other embodiments, the antibodyof choice is a single chain F_(v) fragment (F_(v)) (WO 93/16185).

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanizedor human antibodies. Methods for producing chimeric antibodies are knownin the art, see e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., J Immunol Methods 125:191(1989); and U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397.

Humanized antibodies are derived from antibody molecules generated in anon-human species that bind IL-4 and/or IL-13 wherein one or more CDRstherefrom are inserted into the FR regions from a human immunoglobulinmolecule. Antibodies can be humanized using a variety of techniquesknown in the art including, for example, CDR grafting (EPO 239,400; WO91/09967; and U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089),veneering or resurfacing (EPO 592,106; EPO 519,596; Padlan, MolecularImmunology 28:489 (1991); Studnicka et al., Protein Engineering 7:805(1994); and Roguska et al., Proc Natl Acad Sci USA 91:969 (1994)), andchain shuffling (U.S. Pat. No. 5,565,332).

A humanized antibody has one or more amino acid residues from a sourcethat is non-human. The non-human amino acid residues are often referredto as “import” residues, which are typically taken from an “import”variable domain. Humanization can be essentially performed following themethods of Winter and co-workers (Jones et al., Nature 321:522 (1986);Riechmann et al., Nature 332:323 (1988); and Verhoeyen et al., Science239:1534 (1988)), by substituting non-human CDRs or portions of CDRsequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possible some FRresidues are substituted from analogous sites in rodent antibodies. Theheavy chain constant region and hinge region can be from any class orsubclass to obtain a desired effect, such as a particular effectorfunction.

Often, framework residues in the human framework regions can besubstituted with the corresponding residue from the CDR donor antibodyto alter, and possibly improve, antigen binding. The frameworksubstitutions are identified by methods known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions, see, e.g., U.S. Pat. No. 5,585,089; and Riechmann et al.,Nature 332:323 (1988).

It is further preferable that humanized antibodies retain high affinityfor IL-4 and/or IL-13, and retain or acquire other favorable biologicalproperties. Thus, humanized antibodies are prepared by a process ofanalysis of the parental sequences and various conceptual humanizedproducts using three-dimensional models of the parental and humanizedsequences. Three-dimensional immunoglobulin models are commonlyavailable and are familiar to those skilled in the art. Computerprograms are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of the displays permits analysis ofthe likely role of certain residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind IL-4 and/or IL-13.In that way, FR residues can be selected and combined from the recipientand import sequences so that the desired antibody characteristic, suchas increased affinity for the target antigen, is maximized, although itis the CDR residues that directly and most substantially influence IL-4or IL-13 binding. The CDR regions also can be modified to contain one ormore amino acids that vary from that obtained from the parent antibodyfrom which the CDR was obtained, to provide enhanced or differentproperties of interest, such as binding of greater affinity or greateravidity, for example.

Certain portions of the constant regions of antibody can be manipulatedand changed to provide antibody homologs, derivatives, fragments and thelike with properties different from or better than that observed in theparent antibody. Thus, for example, many IgG4 antibodies form intrachaindisulfide bonds near the hinge region. The intrachain bond candestabilize the parent bivalent molecule forming monovalent moleculescomprising a heavy chain with the associated light chain. Such moleculescan reassociate, but on a random basis.

It was observed that modifying amino acids in the hinge region of IgG4molecules can reduce the likelihood of intrachain bond formation,thereby stabilizing the IgG4 molecule, which will minimize thelikelihood of forming bispecific molecules. That modification can bebeneficial if a therapeutic antibody is an IgG4 molecule as the enhancedstability will minimize the likelihood of having the molecule dissociateduring production and manufacture, as well as in vivo. A monovalentantibody may not have the same effectiveness as the bivalent parentmolecule. For example, when bivalent IgG4 is administered to a patient,the percentage of bivalent IgG4 decays to about 30% over a two-weekperiod. An amino acid substitution at position 228 enhances IgG4stability. The serine that resides at 228 can be replaced with anotheramino acid, such as one of the remaining 19 amino acids. Such a changecan be made particularly with recombinant antibodies wherein the nucleicacid coding sequence can be mutated to yield a replacement amino acid atposition 228. For example, the S can be replaced with a proline.

Another set of amino acids suitable for modification include amino acidsin the area of the hinge which impact binding of a molecule containing aheavy chain with binding to the F_(c) receptor and internalization ofbound antibody. Such amino acids include, in IgG1 molecules, residuesfrom about 233 to about 237 (Glu-Leu-Leu-Gly-Gly); (SEQ ID NO:49) fromabout 252 to about 256 (Met-Ile-Ser-Arg-Thr) (SEQ ID NO:50) and fromabout 318 (Glu) to about 331 (Pro), including, for example, Lys₃₂₀,Lys₃₂₂ and Pro₃₂₉.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences, see, U.S. Pat. Nos. 4,444,887 and 4,716,111; and WO 98/46645,WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO91/10741. The techniques of Cole et al. and Boerder et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985); andBoerner et al., J Immunol 147:86 (1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichalso express certain human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region and diversityregion may be introduced into mouse embryonic stem cells, in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of the human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies, see, e.g., Jakobovitis et al.,Proc Natl Acad Sci USA 90:2551 (1993); Jakobovitis et al., Nature362:255 (1993); Bruggermann et al., Year in Immunol 7:33 (1993); andDuchosal et al., Nature 355:258 (1992)).

The transgenic mice are immunized in the normal fashion with IL-4 orIL-13 cytokine, e.g., all or a portion of IL-4 or IL-13 Monoclonalantibodies directed against IL-4 and IL-13 can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview, see Lonberg et al., Int Rev Immunol13:65-93 (1995). For a discussion of producing human antibodies andhuman monoclonal antibodies and protocols for producing such antibodies,see, e.g., WO 98/24893; WO 92/01047; WO 96/34096; and WO 96/33735; EPONo. 0 598 877; and U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425;5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and5,939,598. In addition, companies such as Amgen (Fremont, Calif.),Genpharm (San Jose, Calif.) and Medarex, Inc. (Princeton, N.J.) can beengaged to provide human antibodies directed against IL-4 and/or IL-13using technology similar to that described above.

Also, human mAbs could be made by immunizing mice transplanted withhuman peripheral blood leukocytes, splenocytes or bone marrow (e.g.,trioma technique of XTL Biopharmaceuticals, Israel). Completely humanantibodies which recognize a selected epitope can be generated using atechnique referred to as “guided selection.” In that approach, aselected non-human monoclonal antibody, e.g., a mouse antibody is usedto guide the selection of a completely human antibody recognizing thesame epitope (Jespers et al., Bio/technology 12:899 (1988)).

When using recombinant techniques, the antibody variant can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody variant is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, maybe removed, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is exposed to sodium acetate (pH 3.5) and EDTA. Celldebris can be removed by centrifugation. Where the antibody variant issecreted into the medium, supernatant from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedto inhibit proteolysis, and antibiotics may be included to preventgrowth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis and affinity chromatography. The suitability of protein A orprotein G as an affinity ligand depends on the species and isotype ofany immunoglobulin F_(e) domain that is present in the antibody variant.Protein A can be used to purify antibodies that are based on human IgG1,IgG2 or IgG4 heavy chains (Lindmark et al., J Immunol Meth 62:1 (1983)).Protein G can be used for mouse isotypes and for human IgG3 (Guss etal., EMBO J. 5:1567 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices, such as controlled pore glass orpoly(styrenedivinyl)benzene, allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodyvariant comprises a C_(H3) domain, the Bakerbond ABX™ resin (JT Baker;Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification, such as fractionation on an ion-exchange column,ethanol precipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin agarose chromatography on an anion or cationexchange resin (such as a polyaspartic acid column), chromatofocusing,SDS-PAGE and ammonium sulfate precipitation are also available,depending on the antibody or variant to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody or variant of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH of between about 2.5-4.5, preferably performed at low saltconcentrations (e.g., from about 0-0.25 M salt).

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies can be monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In a preferred embodiment, the bispecific antibody, fragmentthereof and so on has binding specificities directed towards IL-4 andIL-13.

Methods for making bispecific antibodies are well known. Traditionally,the recombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy chain/light chain pairs, wherethe two heavy chains have different specificities (Milstein et al.,Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, the hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829 and in Trauneckeret al., EMBO J. 10:3655 (1991). Other methods for making bispecificantibodies are provided in, for example, Kufer et al., Trends Biotech22:238-244, 2004.

Antibody variable domains with the desired binding specificities can befused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H2), and C_(H3) regions. It may have thefirst heavy chain constant region (C_(H1)) containing the site necessaryfor light chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth Enzym 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980).It is contemplated that the antibodies may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioester bond. Examplesof suitable reagents for that purpose include iminothiolate andmethyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S.Pat. No. 4,676,980.

In addition, one can generate single-domain antibodies to IL-4 and/orIL-13. Examples of that technology have been described in WO9425591 forantibodies derived from Camelidae heavy chain Ig, as well as inUS20030130496 describing the isolation of single domain fully humanantibodies from phage libraries.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423 (1988);Huston et al., Proc Natl Acad Sci USA 85:5879 (1988); and Ward, et al.,Nature 334:544 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the F_(v) region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional F_(v) fragments in E. coli may also be used (Skerra et al.,Science 242:1038 (1988)).

The instant invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide. Fused or conjugated antibodies of thepresent invention may be used for ease in purification, sec e.g., WO93/21232; EP 439,095; Naramura et al., Immunol Lett 39:91 (1994); U.S.Pat. No. 5,474,981; Gillies et al., Proc Natl Acad Sci USA 89:1428(1992); and Fell et al., J Immunol 146:2446 (1991). The marker aminoacid sequence can be a hexa-histidine peptide, such as the tag providedin a pQE vector (QIAGEN, Inc., Chatsworth, Calif.), among others, manyof which are commercially available, Gentz et al., Proc Natl Acad SciUSA 86:821 (1989). Other peptide tags useful for purification include,but are not limited to, the “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell37:767 (1984)) and the “flag” tag.

One can also create a single peptide chain binding molecules in whichthe heavy and light chain F_(v) regions are connected. Single chainantibodies (“scF_(v)”) and the method of their construction aredescribed in, for example, U.S. Pat. No. 4,946,778. Alternatively,F_(ab) can be constructed and expressed by similar means. All of thewholly and partially human antibodies can be less immunogenic thanwholly murine monoclonal antibodies, and the fragments and single chainantibodies also can be less immunogenic.

Antibodies or antibody fragments can be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature 348:552 (1990). Clarkson et al., Nature 352:624 (1991) and Markset al., J Mol Biol 222:581 (1991) describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Subsequentpublications describe the production of high affinity (nM range) humanantibodies by chain shuffling (Marks et al., Bio/Technology 10:779(1992)), as well as combinatorial infection and in vivo recombination asa strategy for constructing very large phage libraries (Waterhouse etal., Nucl Acids Res 21:2265 (1993)). Thus, the techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

Candidate anti-IL-4 and/or IL-13 antibodies are tested by enzyme-linkedimmunosorbent assay (ELISA), FACS, Western immunoblotting or otherimmunochemical techniques as known in the art.

To determine whether a particular antibody homolog binds to human IL-4and/or IL-13, any conventional binding assay may be used. Useful IL-4and IL-13 binding assays include FACS analysis, ELISA assays, SurfacePlasmon Resonance (Biacore), radioimmunoassays and the like, whichdetect binding of antibody, and functions resulting therefrom, to humanIL-4 and/or IL-13. Full-length and soluble forms of human IL-4 and IL-13taught herein are useful in such assays. The binding of an antibody orhomolog to IL-4 and/or IL-13, or to soluble fragments thereof, mayconveniently be detected through the use of a second antibody specificfor immunoglobulins of the species from which the antibody or homolog isderived.

To determine whether a particular antibody or homolog does or does notsignificantly block binding to IL-4 and/or IL-13, any suitablecompetition assay may be used. Useful assays include, for example, ELISAassays, FACS assays, radioimmunoassays and the like that quantify theability of the antibody or homolog to compete with IL-4 and/or IL-13.Preferably, the ability of ligand to block binding of labeled human IL-4and/or IL-13 to immobilized antibody or homolog is measured.

Antibodies of the instant invention may be described or specified interms of the epitope(s) or portion(s) of IL-4 and/or IL-13 to which theantibody recognizes or specifically binds. The epitope(s) or polypeptideportion(s) may be specified as described herein, e.g., by N-terminal andC-terminal positions, by size in contiguous amino acid residues,conformational epitopes and so on.

Antibodies of the instant invention may also be described or specifiedin terms of cross-reactivity. Antibodies that bind IL-4 and/or IL-13polypeptides, which have at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 65%, at least 60%, atleast 55%, and at least 50% identity (as calculated using methods knownin the art and described herein) to IL-4 and/or IL-13 are also includedin the instant invention.

Antibodies of the instant invention also may be described or specifiedin terms of binding affinity to IL-4 and/or IL-13. Anti-IL-4 and/oranti-IL-13 antibodies may bind with a K_(D) of less than about 10⁻⁷ M,less than about 10⁻⁶ M, or less than about 10⁻⁵ M. Higher bindingaffinities in an antibody of interest can be beneficial, such as thosewith an equilibrium dissociation constant or K_(D) of from about 10⁻⁸ toabout 10⁻¹⁵ M, from about 10⁻⁸ to about 10⁻¹² M, from about 10⁻⁹ toabout 10⁻¹¹ M, or from about 10⁻⁸ to about 10⁻¹⁰ M. The invention alsoprovides antibodies that competitively inhibit binding of an antibody toan epitope of the invention as determined by any method known in the artfor determining competitive binding, for example, the immunoassaysdescribed herein. In preferred embodiments, the antibody competitivelyinhibits binding to the epitope by at least 95%, at least 90%, at least85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least50%.

The instant invention also includes conjugates comprising an antibody ofinterest. The conjugates comprise two primary components, an antibody ofinterest and a second component, which may be a cell-binding agent, acytotoxic agent and so on.

As used herein, the term “cell-binding agent” refers to an agent thatspecifically recognizes and binds to a molecule on the cell surface.Thus, the cell-binding agent can be a CD antigen, a pathogen antigen,such as a virus antigen, a differentiation antigen, a cancer antigen, acell-specific antigen, a tissue-specific antigen, an Ig or Ig-likemolecule and so on.

Cell-binding agents may be of any type as presently known, or thatbecome known, and includes peptides, non-peptides, saccharides, nucleicacids, ligands, receptors and so on, or combinations thereof. Thecell-binding agent may be any compound that can bind a cell, either in aspecific or non-specific manner. Generally, the agent can be an antibody(especially monoclonal antibodies), lymphokines, hormones, growthfactors, vitamins, nutrient-transport molecules (such as transferrin),or any other cell-binding molecule or substance.

Other examples of cell-binding agents that can be used include:polyclonal antibodies; monoclonal antibodies; and fragments ofantibodies such as F_(ab), F_(ab′), F_((ab′)2) and F_(v) (Parham, J.Immunol. 131:2895-2902 (1983); Spring et al., J. Immunol. 113:470-478(1974); and Nisonoff et al., Arch. Biochem. Biophys. 89: 230-244(1960)).

The second component also can be a cytotoxic agent. The term “cytotoxicagent” as used herein refers to a substance that reduces or blocks thefunction, or growth, of cells and/or causes destruction of cells. Thus,the cytotoxic agent can be a taxol, a maytansinoid, such as DM1 or DM4,CC-1065 or a CC-1065 analog, a ricin, mitomycin C and so on. In someembodiments, the cytotoxic agent, as with any binding agent of aconjugate of the instant invention is covalently attached, directly orvia a cleavable or non-cleavable linker, to an antibody of interest.

Examples of suitable maytansinoids include maytansinol and maytansinolanalogs. Maytansinoids inhibit microtubule formation and are highlytoxic to mammalian cells.

Examples of suitable maytansinol analogues include those having amodified aromatic ring and those having modifications at otherpositions. Such suitable maytansinoids are disclosed in U.S. Pat. Nos.4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348;4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Examples of suitable analogues of maytansinol having a modified aromaticring include: (1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared, forexample, by LAH reduction of ansamytocin P2); (2) C-20-hydroxy (orC-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016)(prepared, for example, by demethylation using Streptomyces orActinomyces or dechlorination using lithium aluminum hydride (LAH)); and(3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Examples of suitable analogues of maytansinol having modifications ofother positions include: (1) C-9-SH (U.S. Pat. No. 4,424,219) (preparedby the reaction of maytansinol with H₂S or P₂S₅); (2) C-14-alkoxymethyl(demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598); (3) C-14-hydroxymethyl oracyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No. 4,450,254) (prepared fromNocardia); (4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (preparedby the conversion of maytansinol by Streptomyces); (5) C-15-methoxy(U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewianudiflora); (6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348)(prepared by the demethylation of maytansinol by Streptomyces); and (7)4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titaniumtrichloride/LAH reduction of maytansinol).

The cytotoxic conjugates may be prepared by in vitro methods. To link acytotoxic agent, drug or prodrug to the antibody, commonly, a linkinggroup is used. Suitable linking groups are known in the art and includedisulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. For example,conjugates can be constructed using a disulfide exchange reaction or byforming a thioether bond between an antibody of interest and the drug orprodrug.

As discussed above, the instant invention provides isolated nucleic acidsequences encoding an antibody or functional fragment or variant thereofas disclosed herein, vector constructs comprising a nucleotide sequenceencoding the IL-4 and/or IL-13-binding portion of the antibody orfunctional fragment thereof of the present invention, host cellscomprising such a vector, and recombinant techniques for the productionof the polypeptide.

The vector normally contains components known in the art and generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker or selectiongenes, sequences facilitating and/or enhancing translation, an enhancerelement and so on. Thus, the expression vectors include a nucleotidesequence operably linked to such suitable transcriptional ortranslational regulatory nucleotide sequences such as those derived frommammalian, microbial, viral or insect genes. Examples of additionalregulatory sequences include operators, mRNA ribosomal binding sites,and/or other appropriate sequences which control transcription andtranslation, such as initiation and termination thereof. Nucleotidesequences are “operably linked” when the regulatory sequencefunctionally relates to the nucleotide sequence for the appropriatepolypeptide. Thus, a promoter nucleotide sequence is operably linked to,e.g., the antibody heavy chain sequence if the promoter nucleotidesequence controls the transcription of that nucleotide sequence.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with antibody heavy and/or light chain sequencescan be incorporated into expression vectors. For example, a nucleotidesequence for a signal peptide (secretory leader) may be fused in-frameto the polypeptide sequence so that the antibody is secreted to theperiplasmic space or into the medium. A signal peptide that isfunctional in the intended host cells enhances extracellular secretionof the appropriate antibody or portion thereof. The signal peptide maybe cleaved from the polypeptide on secretion of antibody from the cell.Examples of such secretory signals are well known and include, e.g.,those described in U.S. Pat. Nos. 5,698,435; 5,698,417; and 6,204,023.

The vector may be a plasmid, a single-stranded or double-stranded viralvector, a single-stranded or double-stranded RNA or DNA phage vector, aphagemid, a cosmid or any other carrier of a transgene of interest. Suchvectors may be introduced into cells as polynucleotides by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors also may be introduced into cells aspackaged or encapsulated virus by well known techniques for infectionand transduction. Viral vectors may be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells and using plural vectorscarrying the various virus components necessary to produce a particle.Cell-free translation systems may also be employed to produce theprotein using RNAs derived from the present DNA constructs (see, e.g.,WO 86/05807 and WO 89/01036; and U.S. Pat. No. 5,122,464).

The antibodies of the present invention can be expressed from anysuitable host cell. Examples of host cells useful in the instantinvention include prokaryotic, yeast or higher eukaryotic cells andinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, Serratia, and Shigella, as well as Bacilli, Pseudomonas andStreptomyces) transformed with recombinant bacteriophage DNA, plasmidDNA or cosmid DNA expression vectors containing the antibody codingsequences of interest; yeast (e.g., Saccharomyces, Pichia,Actinomycetes, Kluyveromyces, Schizosaccharomyces, Candida, Trichoderma,Neurospora, and filamentous fungi, such as Neurospora, Penicillium,Tolypocladium and Aspergillus) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,Baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; or tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293 or 3T3 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; or the vaccinia virus 7.5K promoter).

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids, such as pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec,Madison, Wis.), pET (Novagen, Madison, Wis.) and the pRSET (Invitrogen,Carlsbad, Calif.) series of vectors (Studier, J Mol Biol 219:37 (1991);and Schoepfer, Gene 124:83 (1993)). Promoter sequences commonly used forrecombinant prokaryotic host cell expression vectors include T7,(Rosenberg et al., Gene 56:125 (1987)), β-lactamase (penicillinase),lactose promoter system (Chang et al., Nature 275:615 (1978); andGoeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter system(Goeddel et al., Nucl Acids Res 8:4057 (1980)), and tac promoter(Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory (1990)).

Yeast vectors will often contain an origin of replication sequence, suchas from a 2μ yeast plasmid, an autonomously replicating sequence (ARS),a promoter region, sequences for polyadenylation, sequences fortranscription termination and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J BiolChem 255:2073 (1980)) or other glycolytic enzymes (Holland et al.,Biochem 17:4900 (1978)) such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Fleer et al., Gene 107:285 (1991).Other suitable promoters and vectors for yeast and yeast transformationprotocols are well known in the art. Yeast transformation protocols arewell known. One such protocol is described by Hinnen et al., Proc NatlAcad Sci 75:1929 (1978), which selects for Trp⁺ transformants in aselective medium.

Any eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells (Luckow et al., Bio/Technology 6:47 (1988); Miller et al.,Genetic Engineering, Setlow et al., eds., vol. 8, pp. 277-9, PlenumPublishing (1986); and Maeda et al., Nature 315:592 (1985)). Forexample, Baculovirus systems may be used for production of heterologousproteins. In an insect system, Autographa califormica nuclearpolyhedrosis virus (AcNPV) may be used as a vector to express foreigngenes. The virus grows in Spodoptera frugiperda cells. The antibodycoding sequence may be cloned under control of an AcNPV promoter (forexample the polyhedrin promoter). Other hosts that have been identifiedinclude Aedes, Drosophila melanogaster and Bombyx mori. A variety ofviral strains for transfection are publicly available, e.g., the L-1variant of AcNPV and the Bm-5 strain of Bombyx mori NPV. Moreover, plantcell cultures of cotton, corn, potato, soybean, petunia, tomato, andtobacco and also be utilized as hosts as known in the art.

Vertebrate cells and propagation of vertebrate cells in culture (tissueculture) can be a routine procedure, although fastidious cell lines doexist which require, for example, a specialized medium with uniquefactors, feeder cells and so on, see Tissue Culture, Kruse et al., eds.,Academic Press (1973). Examples of useful mammal host cell lines aremonkey kidney; human embryonic kidney line; baby hamster kidney cells;Chinese hamster ovary cells/−DHFR(CHO, Urlaub et al., Proc Natl Acad SciUSA 77:4216 (1980)); mouse sertoli cells; human cervical carcinoma cells(for example, HeLa); canine kidney cells; human lung cells; human livercells; mouse mammary tumor; and NSO cells.

Host cells are transformed with vectors for antibody production andcultured in conventional nutrient medium containing growth factors,vitamins, minerals and so on, as well as inducers appropriate for thecells and vectors used. Commonly used promoter sequences and enhancersequences are derived from polyoma virus, Adenovirus 2, Simian virus 40(SV40) and human cytomegalovirus (CMV). DNA sequences derived from theSV40 viral genome may be used to provide other genetic elements forexpression of a structural gene sequence in a mammalian host cell, e.g.,SV40 origin, early and late promoter, enhancer, splice andpolyadenylation sites. Viral early and late promoters are particularlyuseful because both are easily obtained from a viral genome as afragment which may also contain a viral origin of replication. Exemplaryexpression vectors for use in mammalian host cells are commerciallyavailable.

Commercially available medium such as Ham's F10, Minimal EssentialMedium (MEM), RPMI-1640 and Dulbecco's Modified Eagle's Medium (DMEM)are suitable for culturing host cells. In addition, any of the mediadescribed in Ham et al., Meth Enzymol 58:44 (1979) and Barnes et al.,Anal Biochem 102:255 (1980), and in U.S. Pat. No. 4,767,704; 4,657,866;4,560,655; 5,122,469; 5,712,163; or 6,048,728 may be used as a culturemedium for the host cells. Any of those media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin or epidermal growth factor), salts (such as chlorides, suchas sodium, calcium or magnesium chloride; and phosphates), buffers (suchas HEPES), nucleotides (such as adenosine and thymidine), antibiotics,trace elements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range) and glucose or an equivalentenergy source. Any other necessary supplements may be included atappropriate concentrations, as a design choice. The culture conditions,such as temperature, pH and the like, are as known in the artappropriate for the cell and to enable the desired expression of thetransgene.

The polynucleotides of interest may be obtained, and the nucleotidesequence of the polynucleotides determined, by any method known in theart. For example, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,Bio/Techniques 17:242 (1994)) and then amplifying the ligatedoligonucleotides, for example, by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid of a cell expressing same. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be obtained from a suitable source, such as alibrary, which may be one specific for antibody-producing cells, such ashybridoma cells selected to express an antibody of the invention.Suitable primers can be configured for PCR amplification. Amplifiednucleic acids generated by PCR may then be cloned into replicablecloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody are determined, the nucleotide sequence of the antibody maybe manipulated to obtain the equivalents of interest described hereinusing methods known in the art for manipulating nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR etc.(see, for example, Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory (1990); and Ausubel etal., eds., Current Protocols in Molecular Biology, John Wiley & Sons(1998) to generate antibodies having a different amino acid sequence,for example, to create amino acid substitutions, deletions and/orinsertions.

The amino acid sequence of the heavy and/or light chain variable domainmay be inspected to identify the sequences of the CDRs by well knownmethods, e.g., by comparison to known amino acid sequences of otherheavy and light chain variable regions to determine the regions ofsequence hypervariability. Using routine recombinant DNA techniques, oneor more of the CDRs may be inserted within framework regions, e.g., intohuman framework regions to humanize a non-human antibody, as describedsupra. The polynucleotide of interest generated by the combination ofthe framework regions and one or more CDRs encodes an antibody thatspecifically binds IL-4 and/or IL-13, or at least the ED domain thereof.For example, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds.

The antibodies or antibody fragments of the invention can be used todetect IL-4 and/or IL-13, and hence cells expressing IL-4 and/or IL-13,in a biological sample in vitro or in vivo. In one embodiment, theanti-IL-4 and/or IL-13 antibody of the invention is used to determinethe presence and the level of IL-4 and/or IL-13 in a tissue or in cellsderived from the tissue. The levels of IL-4 and/or IL-13 in the tissueor biopsy can be determined, for example, in an immunoassay with theantibodies or antibody fragments of the invention. The tissue or biopsythereof can be frozen or fixed. The same or other methods can be used todetermine other properties of IL-4 and/or IL-13, such as the levelthereof, cellular localization, mRNA levels, mutations thereof and soon.

The above-described method can be used, for example, to diagnose acancer in a subject known to be or suspected to have a cancer, whereinthe level of IL-4 and/or IL-13 measured in said patient is compared withthat of a normal reference subject or standard. The assay of interestalso can be used to diagnose arthritis or other autoimmune diseasescharacterized by B cell infiltration and concentration, along withdevelopment of differentiated lymphoid tissue.

The instant invention further provides for monoclonal antibodies,humanized antibodies and epitope-binding fragments thereof that arefurther labeled for use in research or diagnostic applications. In someembodiments, the label is a radiolabel, a fluorophore, a chromophore, animaging agent or a metal ion.

A method for diagnosis is also provided in which said labeled antibodiesor epitope-binding fragments thereof are administered to a subjectsuspected of having a cancer, arthritis, autoimmune diseases or otherIL-4 and/or IL-13 mediated disease, and the distribution of the labelwithin the body of the subject is measured or monitored.

The antibody and fragments thereof of the instant invention may be usedas affinity purification agents. In that process, the antibodies areimmobilized on a solid phase, such as a dextran or agarose resin orfilter paper, using methods known in the art. The immobilized antibodyis contacted with a sample containing IL-4 and/or IL-13 or cellscarrying same to be purified, and thereafter the support is washed witha suitable solvent that will remove substantially all the material inthe sample except the IL-4 and/or IL-13 or cell to be purified, which isbound to the immobilized antibody of interest. Finally, the support iswashed with another suitable solvent, such as glycine buffer, pH 5.0that will release the IL-4 and/or IL-13 or cell from the antibody ofinterest.

For diagnostic applications, the antibody of interest typically will belabeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories: (a)radioisotopes, such as ³⁶S, ¹⁴C, ¹²⁵I, ³H and ¹³¹I (The antibody can belabeled with the radioisotope using a techniques described in CurrentProtocols in Immunology, vol. 12, Coligen et al., ed.,Wiley-Interscience, New York (1991), for example, and radioactivity canbe measured using scintillation counting); (b) fluorescent labels, suchas rare earth chelates (europium chelates), fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, lissamine,phycoerythrin and Texas Red, the fluorescent labels can be conjugated tothe antibody using a technique disclosed in Current Protocols inImmunology, supra, for example, where fluorescence can be quantifiedusing a fluorimeter; and (c) various enzyme substrate labels areavailable (U.S. Pat. No. 4,275,149 provides a review), the enzymegenerally catalyzes a chemical alteration of the chromogenic substratewhich can be measured using various techniques, for example, the enzymemay catalyze a color change in a substrate, which can be measuredspectrophotometrically, or the enzyme may alter the fluorescence orchemiluminescence of the substrate. Techniques for quantifying a changein fluorescence are known, for example, using a luminometer, or thelabel donates energy to a fluorescent acceptor. Examples of enzymaticlabels include luciferases (e.g., firefly luciferase and bacterialluciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase,such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Meth Enz, ed. Langone & Van Vunakis, Academic Press, New York, 73(1981).

When such labels are used, suitable substrates are available, such as:(i) for horseradish peroxidase with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.,orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB)); (ii) for alkaline phosphatase (AP) withp-nitrophenyl phosphate as the chromogenic substrate; and (iii)β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate such as4-methylumbelliferyl-β-D-galactosidase.

Other enzyme-substrate combinations are available to those skilled inthe art. For a general review, see U.S. Pat. Nos. 4,275,149 and4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Forexample, the antibody can be conjugated with biotin and any of thereporters mentioned above can be conjugated with avidin, or vice versa.Biotin binds selectively to avidin and thus, the label can be conjugatedwith the antibody in that indirect manner. Alternatively, to achieveindirect conjugation of the label, the antibody is conjugated with asmall hapten (e.g., digoxin) and one of the different types of labels orreporters mentioned above is conjugated with an anti-digoxin antibody.Thus, indirect conjugation of the label with the antibody or mutein canbe achieved using a second antibody.

In another embodiment of the invention, the antibody need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody, another form of a second antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample for binding with a limited amount ofantibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies may conveniently be separated from thestandard and test sample which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, determinant or epitope, ofthe target to be detected. In a sandwich assay, the test sample to beanalyzed is bound by a first antibody which is immobilized directly orindirectly on a solid support, and thereafter a second antibody directlyor indirectly labeled binds to the bound test sample, thus forming aninsoluble three-part complex, see e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody or other suitable member of the binding pair (antibody/antigen,receptor/ligand, enzyme/substrate, for example) that is labeled with adetectable moiety (indirect sandwich assay). For example, one type ofsandwich assay is an ELISA assay, in which case the detectable moiety isan enzyme.

The instant invention also includes kits, e.g., comprising an antibody,fragment thereof, homolog, derivative thereof and so on, such as alabeled or cytotoxic conjugate, and instructions for the use of theantibody, conjugate for killing particular cell types and so on. Theinstructions may include directions for using the antibody, conjugateand so on in vitro, in vivo or ex vivo. The antibody can be in liquidform or as a solid, generally lyophilized. The kit can contain suitableother reagents, such as a buffer, a reconstituting solution and othernecessary ingredients for the intended use. A packaged combination ofreagents in predetermined amounts with instructions for use thereof,such as for a therapeutic use of for performing a diagnostic assay iscontemplated. Where the antibody is labeled, such as with an enzyme, thekit can include substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied toprovide for concentrates of a solution of a reagent, which provides userflexibility, economy of space, economy of reagents and so on. Thereagents may be provided as dry powders, usually lyophilized, includingexcipients, which on dissolution provide a reagent solution having theappropriate concentration.

The antibodies of the present invention may be used to treat a mammal.In one embodiment, the antibody or equivalent of interest isadministered to a nonhuman mammal for the purposes of obtainingpreclinical data, for example. Exemplary nonhuman mammals to be treatedinclude nonhuman primates, dogs, cats, rodents and other mammals inwhich preclinical studies are performed. Such mammals may be establishedanimal models for a disease to be treated with the antibody, or may beused to study toxicity of the antibody of interest. In each of thoseembodiments, dose escalation studies may be performed in the mammal.

An antibody, with or without a second component, such as a therapeuticmoiety conjugated to same, administered alone or in combination withcytotoxic factor(s) can be used as a therapeutic. The present inventionis directed to antibody-based therapies which involve administeringantibodies of the invention to an animal, a mammal, or a human, fortreating a IL-4 and/or IL-13 mediated disease, disorder or condition.

The term “treatment” as used in the present invention refers to boththerapeutic treatment and prophylactic or preventative measures. Itrefers to preventing, curing, reversing, attenuating, alleviating,minimizing, suppressing or halting the deleterious effects of a diseasestate, disease progression, disease causative agent (e.g., bacteria orviruses) or other abnormal condition.

Thus the invention also includes polyvalent antibodies, includingbispecific anti-IL-4/IL-13 antibodies, having attached theretodiagnostically or therapeutically functional effector molecules, atomsor other species. For example, the antibody may have a radioactivediagnostic label or radioactive cytotoxic atom or metal or cytotoxicspecies, e.g. ricin chain, attached thereto for in vivo diagnosis ortherapy of cancer.

Moreover, the antibodies according to the invention may be used inimmunoassays, in purification methods and in other methods in whichimmunoglobulins or fragments thereof are used. Such uses are well-knownin the art.

Accordingly, the invention also provides compositions comprising theanti-IL-13 and/or anti-IL-4 antibodies or fragments thereof according tothe invention, conveniently in combination with a pharmaceuticallyacceptable carrier, diluent or excipient which are conventional in theart.

The term “pharmaceutical composition” as used in the present inventionrefers to formulations of various preparations. The formulationscontaining therapeutically effective amounts of the polyvalentantibodies are sterile liquid solutions, liquid suspensions orlyophilized versions and optionally contain stabilizers or excipients.

The term “disorder” as used in the present invention refers to anycondition that would benefit from treatment with the antibody of thepresent invention. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal, andin particular humans, to the disorder in question. Non-limiting examplesof disorders to be treated herein include cancers, inflammation,autoimmune diseases, infections, cardiovascular diseases, respiratorydiseases, neurological diseases and metabolic diseases.

The antibodies of the present invention may be used to treat, suppressor prevent disease, such as an allergic disease, a Th2-mediated disease,IL-13-mediated disease, IL-4-mediated disease, and/orIL-4/IL-13-mediated disease. Examples of such diseases include,Hodgkin's disease, asthma, allergic asthma, atopic dermatitis, atopicallergy, ulcerative colitis, scleroderma, allergic rhinitis, COPD3idiopathic pulmonary fibrosis, chronic graft rejection,bleomycin-induced pulmonary fibrosis, radiation-induced pulmonaryfibrosis, pulmonary granuloma, progressive systemic sclerosis,schistosomiasis, hepatic fibrosis, renal cancer, Burkitt lymphoma,Hodgkins disease, non˜Hodgkins disease, Sezary syndrome, asthma, septicarthritis, dermatitis herpetiformis, chronic idiopathic urticaria,ulcerative colitis, scleroderma, hypertrophic scarring, Whipple'sDisease, benign prostate hyperplasia, a lung disorder in which IL-4receptor plays a role, condition in which IL-4 receptor-mediatedepithelial barrier disruption plays a role, a disorder of the digestivesystem in which IL-4 receptor plays a role, an allergic reaction to amedication, Kawasaki disease, sickle cell disease, Churg-Strausssyndrome, Grave's disease, pre-eclampsia, Sjogren's syndrome, autoimmunelymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett'sesophagus, autoimmune uveitis, tuberculosis, cystic fibrosis, allergicbronchopulmonary mycosis, chronic obstructive pulmonary disease,bleomycin-induced pneumopathy and fibrosis, pulmonary alveolarproteinosis, adull respiratory distress syndrome, sarcoidosis, hyper IgEsyndrome, idiopathic hypercosinophil syndrome, an autoimmune blisteringdisease, pemphigus vulgaris, bullous pemphigoid, myasthenia gravis,chronic fatigue syndrome, nephrosis).

The term “allergic disease” refers to a pathological condition in whicha patient is hypersensitized to and mounts an immunologic reactionagainst a substance that is normally nonimmunogenic. Allergic disease isgenerally characterized by activation of mast cells by IgE resulting inan inflammatory response (e.g. local response, systemic response) thatcan result in symptoms as benign as a runny nose, to life-threateninganaphylactic shock and death. Examples of allergic disease include, butare not limited to, allergic rhinitis (e.g., hay fever), asthma (e.g.,allergic asthma), allergic dermatitis (e.g., eczema), contactdermatitis, food allergy and urticaria (hives).

As used herein “Th2-mediated disease” refers to a disease in whichpathology is produced (in whole or in part) by an immune response(Th2-type immune response) that is regulated by CD4⁺ Th2 T lymphocytes,which characteristically produce IL-4, IL-5, IL-9 and IL-13. A Th2-typeimmune response is associated with the production of certain cytokines(e.g., IL-4, IL-13) and of certain classes of antibodies (e.g., IgE),and is associate with humoral immunity. Th2-mediated diseases arecharacterized by the presence of elevated levels of Th2 cytokines (e.g.,IL-4, IL-13) and/or certain classes of antibodies (e.g., IgE) andinclude, for example, allergic disease (e.g., allergic rhinitis, atopicdermatitis, asthma (e.g., atopic asthma), allergic airways disease(AAD), anaphylactic shock, conjunctivitis), autoimmune disordersassociated with elevated levels of IL-4 and/or IL-13 (e.g., rheumatoidarthritis, host-versus-graft disease, renal disease (e.g., nephriticsyndrome, lupus nephritis)), and infections associated with elevatedlevels of IL-4 and/or IL-13 (e.g., viral, parasitic, fungal (e.g., C.albicans) infection). Certain cancers are associated with elevatedlevels of IL-4 and/or IL-13 or associated with IL-4-induced and/orIL-13-induced cancer cell proliferation (e.g., B cell lymphoma, T celllymphoma, multiple myeloma, head and neck cancer, breast cancer andovarian cancer). These cancers can be treated, suppressed or preventedusing the Ii gaud of the invention.

The term “cancer” as used in the present invention refers to ordescribes the physiological condition in mammals, in particular humans,which is typically characterized by unregulated cell growth. Examples ofcancer include but are not limited to, carcinoma, lymphoma, blastoma,sarcoma, and leukemia.

The term “autoimmune disease” as used in the present invention refers toa non-malignant disease or disorder arising from and directed against anindividual's own tissues. Examples of autoimmune diseases or disordersinclude, but are not limited to, inflammatory responses such asinflammatory skin diseases including psoriasis and dermatitis; allergicconditions such as eczema and asthma; other conditions involvinginfiltration of T cells and chronic inflammatory responses;atherosclerosis; diabetes mellitus (e.g. Type I diabetes mellitus orinsulin dependent diabetes mellitis); multiple sclerosis and centralnervous system (CNS) inflammatory disorder.

The antibodies of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. Theantibodies can be used in combination therapy with existing IL-13therapeutics (e.g. existing IL-13 agents such as anti-IL-13Rα1, IL-4/13Trap, anti-IL-13) plus anti-IL-4 antibody and existing IL-4 agents (forexample, anti-IL-4R, IL-4 Mutein, IL-4/13 Trap) plus anti-IL-13 antibodyand IL-4 antibodies (for example, WO05/0076990 (CAT), WO03/092610(Regeneron), WO00/64944 (Genetic Inst.) and WO2005/062967 (Tanox)).

The antibodies of the present invention may be administered and/orformulated together with one or more additional therapeutic or activeagents. When a ligand is administered with an additional therapeuticagent, the ligand can be administered before, simultaneously with orsubsequent to administration of the additional agent. Generally, theligand and additional agent are administered in a manner that providesan overlap of therapeutic effect. Additional agents that can beadministered or formulated with the ligand of the invention include, forexample, various immunotherapeutic dings?, such as cylcosporine,methotrexate, adriamycin or cisplatimun, antibiotics, antimycotics,anti-viral agents and immunotoxins. For example, when the antagonist isadministered to prevent, suppress or treat lung inflammation or arespiratory disease (e.g., asthma), it can be administered in conjuctionwith phosphodiesterase inhibitors (e.g., inhibitors of phosphodiesterase4), bronchodilators (e.g., β2-agonists, anticholinergerics,theophylline), short-acting beta-agonists (e.g., albuterol, salbuiamol,bambuterol, fenoter[sigma]1, isoetherine, isoproterenol,leva[iota]buterol, metaproterenol, pirbuterol, terbutaline andtornlate), long-acting beta-agonists (e.g., formoterol and salmeterol),short acting anticholinergics (e.g., ipratropium bromide and oxitropiumbromide), long-acting anticholinergics (e.g., tiotropium), theophylline(e.g. short acting formulation, long acting formulation), inhaledsteroids (e.g., beclomethasone, beclometasone, budesonide, flunisolide,fluticasone propionate and triamcinolone), oral steroids (e.g.,methylprednisolone, prednisolone, prednisolon and prednisone), combinedshort-acting beta-agonists with anticholinergics (e.g.,albuterol/salbutamol/ipratopium, and fenoterol/ipratopium), combinedlong-acting beta-agonists with inhaled steroids (e.g.,salmeterol/fluticasone, and formolerol/budesonide) and mucolytic agents(e.g., erdosteine, acetylcysteine, bromheksin, carbocyslcine,guiafencsin and iodinated glycerol

Other suitable co-therapeutic agents that can be administed withantibody of the present invention to prevent, suppress or treat asthma(e.g., allergic asthma), include a corticosteroid (e.g., beclomethasone,budesonide, fluticasone), cromoglycate, nedocromil, beta-agonist (e.g.,salbutamol, terbutaline, bambuterol, fenoterol, reproterol, tolubuterol,salmeterol, fomtero), zafirlukast, salmeterol, prednisone, prednisolone,theophylline, zileutron, montelukast, and leukotriene modifiers. Theligands of the invention can be coadministered with a variety ofco-therapeutic agents suitable for treating diseases (e.g., a Th-2mediated disease, YL-A-mediatcd disease, IL-13 mediated disease, IL-4mediated disease and cancer), including cytokines,analgesics/antipyretics, antiemetics, and chemotherapeutics.

Antibodies of the invention may be provided in pharmaceuticallyacceptable compositions as known in the art or as described herein. Theterm “physiologically acceptable,” “pharmacologically acceptable” and soon mean approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals and more particularly inhumans.

The anti-IL-4, anti-IL-13 and bispecific anti-IL-4/IL-13 antibodies maybe administered to a mammal and in particular humans, in any acceptablemanner. Methods of introduction include, but are not limited to,parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal,epidural, inhalation and oral routes, and if desired forimmunosuppressive treatment, intralesional administration. Parenteralinfusions include intramuscular, intradermal, intravenous, intraarterialor intraperitoneal administration. The antibodies or compositions may beadministered by any convenient route, for example, by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the therapeutic antibodies or compositions of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Inaddition, the antibody is suitably administered by pulse infusion,particularly with declining doses of the antibody. Preferably the dosingis given by injection, preferably intravenous or subcutaneousinjections, depending, in part, on whether the administration is briefor chronic.

Various other delivery systems are known and can be used to administeran antibody of the present invention, including, e.g., encapsulation inliposomes, microparticles, microcapsules (see Langer, Science 249:1527(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein et al., eds., p. 353-365 (1989); andLopez-Berestein, ibid., p. 317-327) and recombinant cells capable ofexpressing the compound; receptor-mediated endocytosis (see, e.g., Wu etal., J Biol Chem 262:4429 (1987)); construction of a nucleic acid aspart of a retroviral or other vector etc.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmcthacylatc) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nanoparticles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed. (1980).

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. Theantibody may also be administered into the lungs of a patient in theform of a dry powder composition, see e.g., U.S. Pat. No. 6,514,496.

In a specific embodiment, it may be desirable to administer thetherapeutic antibodies or compositions of the invention locally to thearea in need of treatment; that may be achieved by, for example, and notby way of limitation, local infusion, topical application, by injection,by means of a catheter, by means of a suppository or by means of animplant, said implant being of a porous, non-porous or gelatinousmaterial, including membranes, such as sialastic membranes or fibers.Preferably, when administering an antibody of the invention, care istaken to use materials to which the protein does not absorb or adsorb.

In yet another embodiment, the antibody can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,Science 249:1527 (1990); Sefton, CRC Crit. Ref Biomed Eng 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N Engl J Med321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer et al., eds.,CRC Press (1974); Controlled Drug Bioavailability, Drug Product Designand Performance, Smolen et al., eds., Wiley (1984); Ranger et al., JMacromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al.,Science 228:190 (1985); During et al., Ann Neurol 25:351 (1989); andHoward et al., J Neurosurg 71:105 (1989)). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget.

Therapeutic formulations of the polypeptide or antibody may be preparedfor storage as lyophilized formulations or aqueous solutions by mixingthe polypeptide having the desired degree of purity with optional“pharmaceutically acceptable” carriers, diluents, excipients orstabilizers typically employed in the art, i.e., buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants and other miscellaneous additives, see Remington'sPharmaceutical Sciences, 16th ed., Osol, ed. (1980). Such additives aregenerally nontoxic to the recipients at the dosages and concentrationsemployed, hence, the excipients, diluents, carriers and so on arepharmaceutically acceptable.

An “isolated” or “purified” antibody is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourceor medium from which the protein is derived, or substantially free ofchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof an antibody in which the polypeptide/protein is separated fromcellular components of the cells from which same is isolated orrecombinantly produced. Thus, an antibody that is substantially free ofcellular material includes preparations of the antibody having less thanabout 30%, 20%, 10%, 5%, 2.5% or 1%, (by dry weight) of contaminatingprotein. When the antibody is recombinantly produced, it is alsopreferably substantially free of culture medium, i.e., culture mediumrepresents less than about 20%, 10%, 5%, 2.5% or 1% of the volume of theprotein preparation. When antibody is produced by chemical synthesis, itis preferably substantially free of chemical precursors or otherchemicals and reagents, i.e., the antibody of interest is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly, such preparations of the antibodyhave less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemicalprecursors or compounds other than antibody of interest. In a preferredembodiment of the present invention, antibodies are isolated orpurified.

As used herein, the phrase “low to undetectable levels of aggregation”refers to samples containing no more than 5%, no more than 4%, no morethan 3%, no more than 2%, no more than 1% and often no more than 0.5%aggregation, by weight protein, as measured by, for example, highperformance size exclusion chromatography (HPSEC).

As used herein, the term “low to undetectable levels of fragmentation”refers to samples containing equal to or more than 80%, 85%, 90%, 95%,98% or 99%, of the total protein, for example, in a single peak, asdetermined by HPSEC, or in two (2) peaks (heavy chain and light chain)by, for example, reduced capillary gel electrophoresis (rCGE) andcontaining no other single peaks having more than 5%, more than 4%, morethan 3%, more than 2%, more than 1% or more than 0.5% of the totalprotein, each. The rCGE as used herein refers to capillary gelelectrophoresis under reducing conditions sufficient to reduce disulfidebonds in an antibody or antibody-type or derived molecule.

As used herein, the terms “stability” and “stable” in the context of aliquid formulation comprising a Il-4 and/or IL-13 antibody or bindingfragment thereof refer to the resistance of the antibody orantigen-binding fragment thereof in the formulation to thermal andchemical unfolding, aggregation, degradation or fragmentation undergiven manufacture, preparation, transportation and storage conditions.The “stable” formulations of the invention retain biological activityequal to or more than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% under givenmanufacture, preparation, transportation and storage conditions. Thestability of said antibody preparation can be assessed by degrees ofaggregation, degradation or fragmentation by methods known to thoseskilled in the art, including, but not limited to, rCGE, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPSEC,compared to a reference.

The term, “carrier,” refers to a diluent, adjuvant, excipient or vehiclewith which the therapeutic is administered. Such physiological carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a suitablecarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions also can be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. The compositionscan take the form of solutions, suspensions, emulsion, tablets, pills,capsules, powders, sustained-release formulations, depots and the like.The composition can be formulated as a suppository, with traditionalbinders and carriers such as triglycerides. Oral formulations caninclude standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate etc. Examples of suitable carriers are described in“Remington's Pharmaceutical Sciences,” Martin. Such compositions willcontain an effective amount of the antibody, preferably in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the patient. As known in the art, theformulation will be constructed to suit the mode of administration.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. Buffers are preferably present at aconcentration ranging from about 2 mM to about 50 mM. Suitable bufferingagents for use with the instant invention include both organic andinorganic acids, and salts thereof, such as citrate buffers (e.g.,monosodium citrate-disodium citrate mixture, citric acid-trisodiumcitrate mixture, citric acid-monosodium citrate mixture etc.), succinatebuffers (e.g., succinic acid-monosodium succinate mixture, succinicacid-sodium hydroxide mixture, succinic acid-disodium succinate mixtureetc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture,tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxidemixture etc.), fumarate buffers (e.g., fumaric acid-monosodium fumaratemixture, fumaric acid-disodium fumarate mixture, monosodiumfumarate-disodium fumarate mixture etc.), gluconate buffers (e.g.,gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxidemixture, gluconic acid-potassium gluconate mixture etc.), oxalatebuffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodiumhydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactatebuffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodiumhydroxide mixture, lactic acid-potassium lactate mixture etc.) andacetate buffers (e.g., acetic acid-sodium acetate mixture, aceticacid-sodium hydroxide mixture etc.). Phosphate buffers, carbonatebuffers, histidine buffers, trimethylamine salts such as Tris, HEPES andother such known buffers can be used.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, m-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzyaconium halides (e.g., chloride, bromide and iodide),hexamethonium chloride, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are present to ensure physiological isotonicity of liquidcompositions of the instant invention and include polhydric sugaralcohols, preferably trihydric or higher sugar alcohols, such asglycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.Polyhydric alcohols can be present in an amount of between about 0.1% toabout 25%, by weight, preferably 1% to 5% taking into account therelative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols;amino acids, such as arginine, lysine, glycine, glutamine, asparagine,histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamicacid, threonine etc., organic sugars or sugar alcohols, such as lactose,trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol,ribitol, myoinisitol, galactitol, glycerol and the like, includingcyclitols such as inositol; polyethylene glycol; amino acid polymers;sulfur containing reducing agents, such as urea, glutathione, thiocticacid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodiumthiosulfate; low molecular weight polypeptides (i.e., <10 residues);proteins, such as human serum albumin, bovine serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone,saccharides, monosaccharides, such as xylose, mannose, fructose,glucose; disaccharides, such as lactose, maltose and sucrose;trisaccharides such as raffinose; polysaccharides such as dextran and soon. Stabilizers are present in the range from 0.1 to 10,000 w/w per partof active protein.

Additional miscellaneous excipients include bulking agents, (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine or vitamin E) and cosolvents.

The formulation herein also may contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely impact each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules suitably are present in combination in amountsthat are effective for the purpose intended.

As used herein, the term “surfactant” refers to organic substanceshaving amphipathic structures, namely, are composed of groups ofopposing solubility tendencies, typically an oil-soluble hydrocarbonchain and a water-soluble ionic group. Surfactants can be classified,depending on the charge of the surface-active moiety, into anionic,cationic and nonionic surfactants. Surfactants often are used aswetting, emulsifying, solubilizing and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent, as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stresseswithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80 etc.), polyoxamers (184, 188etc.), Pluronic® polyols and polyoxyethylene sorbitan monoethers(TWEEN-20®, TWEEN-80® etc.). Non-ionic surfactants may be present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

As used herein, the term, “inorganic salt,” refers to any compound,containing no carbon that result from replacement of part or all of theacid hydrogen or an acid by a metal or group acting like a metal, andoften are used as a tonicity adjusting compound in pharmaceuticalcompositions and preparations of biological materials. The most commoninorganic salts are NaCl, KCl, NaH₂PO₄ etc.

The instant invention encompasses liquid formulations having stabilityat temperatures found in a commercial refrigerator and freezer found inthe office of a physician or laboratory, such as from about −20° C. toabout 5° C., said stability assessed, for example, by high performancesize exclusion chromatography (HPSEC), for storage purposes, such as forabout 60 days, for about 120 days, for about 180 days, for about a year,for about 2 years or more. The liquid formulations of the presentinvention also exhibit stability, as assessed, for example, by HSPEC, atroom temperatures, for a at least a few hours, such as one hour, twohours or about three hours prior to use.

The term “small molecule” and analogous terms include, but are notlimited to, peptides, peptidomimetics, amino acids, amino acidanalogues, polynucleotides, polynucleotide analogues, nucleotides,nucleotide analogues, organic or inorganic compounds (i.e., includingheterorganic and/or ganometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 5,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 1,000grams per mole, organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such compounds.

Thus, in the case of cancer, the antibodies of the invention may beadministered alone or in combination with other types of cancertreatments, including conventional chemotherapeutic agents (paclitaxel,carboplatin, cisplatin and doxorubicin), anti-EGFR agents (gefitinib,erlotinib and cetuximab), anti-angiogenesis agents (bevacizumab andsunitinib), as well as immunomodulating agents, such as interferon-α andthalidomide.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment, management oramelioration of a disease, disorder, malady and the like associated withaberrant IL-4 and/or IL-13 metabolism and activity.

In addition, the antibodies of the instant invention may be conjugatedto various effector molecules such as heterologous polypeptides, drugs,radionucleotides or toxins, see, e.g., WO 92/08495; WO 91/14438; WO89/12624; U.S. Pat. No. 5,314,995; and EPO 396,387. An antibody orfragment thereof may be conjugated to a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive metal ion (e.g., α emitters, such as, for example,²¹³Bi). A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include paclitaxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicine, doxorubicin,daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol and puromycin and analogs orhomologues thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil and decarbazine), alkylating agents (e.g.,mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin, daunomycin anddoxorubicin), antibiotics (e.g., dactinomycin, actinomycin, bleomycin,mithramycin and anthramycin (AMC)), and anti-mitotic agents (e.g.,vincristine and vinblastine).

Techniques for conjugating such a therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., in Monoclonal Antibodies and CancerTherapy, Reisfeld et al. (eds.), p. 243-56 Alan R. Liss (1985);Hellstrom et al., in Controlled Drug Delivery, 2nd ed., Robinson et al.,eds., p. 623-53, Marcel Dekker (1987); Thorpe, in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al., eds., p.475-506 (1985); Monoclonal Antibodies For Cancer Detection and Therapy,Baldwin et al., eds., p. 303-16, Academic Press (1985); and Thorpe, etal., Immunol Rev 62:119 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate,such as a bifunctional antibody, see, e.g., U.S. Pat. No. 4,676,980.

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-α, TNF-β, AIM I (WO 97/33899),AIM II (WO 97/34911), Fas ligand (Takahashi et al., Int Immunol, 6:1567(1994)), VEGF (WO 99/23105); a thrombotic agent; an anti-angiogenicagent, e.g., angiostatin or endostatin; or biological response modifierssuch as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2(IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (GCSF) or othergrowth factors.

The formulations to be used for in vivo administration must be sterile.That can be accomplished, for example, by filtration through sterilefiltration membranes. For example, the liquid formulations of thepresent invention may be sterilized by filtration using a 0.2 μm or a0.22 μm filter.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films or matrices. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethylmethacrylate), poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers (such as injectable microspherescomposed of lactic acid-glycolic acid copolymer) andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. Rational strategies can be devised for stabilization dependingon the mechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, amino acid substitutionand developing specific polymer matrix compositions.

The antibody or variant composition will be formulated, dosed andadministered in a manner consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal or human being treated, the clinicalcondition of the individual patient, the cause of the disorder, the siteof delivery of the agent, the method of administration, the schedulingof administration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody or variant to beadministered will be governed by such considerations, and can be theminimum amount necessary to prevent, ameliorate or treat a IL-4 and/orIL-13 mediated disease, condition or disorder.

The antibody or variant optionally is formulated with one or more agentscurrently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as used hereinbefore or about from 1 to99% of the heretofore employed dosages.

As used herein, the term “effective amount” refers to the amount of atherapy (e.g., a prophylactic or therapeutic agent), which is sufficientto reduce the severity and/or duration of a IL-4 and/or IL-13 mediateddisease, ameliorate one or more symptoms thereof, prevent theadvancement of a IL-4 and/or IL-13 mediated disease or cause regressionof a disease, or which is sufficient to result in the prevention of thedevelopment, recurrence, onset, or progression of a IL-4 and/or IL-13mediated disease or one or more symptoms thereof, or enhance or improvethe prophylactic and/or therapeutic effect(s) of another therapy (e.g.,another therapeutic agent) useful for treating a IL-4 and/or IL-13mediated disease.

The amount of therapeutic antibody or fragment thereof which will beeffective in the use or treatment of a particular disorder or conditionwill depend on the nature of the disorder or condition, and can bedetermined by standard clinical techniques. Where possible, adose-response curve and the pharmaceutical compositions of the inventioncan be first derived in vitro. If a suitable animal model system isavailable, again a dose-response curve can be obtained and used toextrapolate a suitable human dose practicing methods known in the art.However, based on common knowledge of the art, a pharmaceuticalcomposition effective in promoting a diminution of an inflammatoryeffect, for example, may provide a local therapeutic agent concentrationof between about 5 and 20 ng/ml, and, preferably, between about 10 and20 ng/ml.

In a preferred embodiment, an aqueous solution of therapeuticpolypeptide, antibody or fragment thereof can be administered bysubcutaneous injection. Each dose may range from about 0.5 mg to about50 mg per kilogram of body weight, or more preferably, from about 3 mgto about 30 mg per kilogram body weight. The dosage can be ascertainedempirically for the particular disease, patient population, mode ofadministration and so on, practicing pharmaceutical methods known in theart.

The dosing schedule for subcutaneous administration may vary from once aweek to daily depending on a number of clinical factors, including thetype of disease, severity of disease and the sensitivity of the subjectto the therapeutic agent.

The instant invention provides methods for preparing liquid formulationsof the antibody or IL-4 and/or IL-13 binding fragment thereof, saidmethods comprising concentrating a fraction of purified antibody to afinal concentration of about 15 mg/ml, about 20 mg/ml, about 30 mg/ml,about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80mg/ml, about 90 mg/ml, about 100 mg/ml, about 200 mg/ml, about 250mg/ml, about 300 mg/ml or more using, for example, a semi-permeablemembrane with an appropriate molecular weight (mw) cutoff (e.g., 30 kDcutoff for F_((ab′)2) fragments thereof; and 10 kD cutoff for F_(ab)fragments).

In addition, the present invention also encompasses stable liquidformulations of the products of interest that have improved half-life invivo. Thus, the antibody of interest has a half-life in a subject,preferably a human, of greater than 3 days, greater than 7 days, greaterthan 10 days, greater than 15 days, greater than 25 days, greater than30 days, greater than 35 days, greater than 40 days, greater than 45days, greater than 2 months, greater than 3 months, greater than 4months, greater than 5 months or more.

To prolong the serum circulation of an antibody in vivo, varioustechniques can be used. For example, inert polymer molecules, such ashigh molecular weight polyethylene glycol (PEG), can be attached to anantibody with or without a multifunctional linker either throughsite-specific conjugation of the PEG to the N-terminus or to theC-terminus of the antibody or via ε amino groups present on lysineresidues. Linear or branched polymer derivatization that results inminimal loss of biological activity can be used. The degree ofconjugation can be closely monitored by SDS-PAGE and mass spectrometryto ensure proper conjugation of PEG molecules to the antibodies.Unreacted PEG can be separated from antibody-PEG conjugates bysize-exclusion or by ion exchange chromatography. PEG-derivatizedantibodies can be tested for binding activity as well as for in vivoefficacy using methods known to those of skilled in the art, forexample, by immunoassays described herein.

An antibody having an increased half-life in vivo can also be generatedby introducing one or more amino acid modifications (i.e.,substitutions, insertions or deletions) into an IgG constant domain, orF_(c)R binding fragment thereof (such as an F_(e) or hinge F_(e) domainfragment), see, e.g., WO 98/23289; WO 97/34631; and U.S. Pat. No.6,277,375.

Further, an antibody can be conjugated to albumin to make an antibodymore stable in vivo or have a longer half life in vivo. The techniquesare known in the art, see e.g., WO 93/15199, WO 93/15200 and WO01/77137; and EPO 413, 622. The antibody also can be modified, forexample, by glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein and so on.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine or other “caine”anesthetic to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water-freeconcentrate in a sealed container, such as an ampule or sachetindicating the quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampule of sterile water forinjection or saline can be provided, for example, in a kit, so that theingredients may be mixed prior to administration.

The invention also provides that a liquid formulation of the presentinvention is packaged in a sealed container such as an ampule or sachetindicating the quantity of the product of interest. The liquidformulations of the instant invention can be in a sealed containerindicating the quantity and concentration of the antibody or antibodyfragment. The liquid formulation of the instant invention can besupplied in a sealed container with at least 15 mg/ml, 20 mg/ml, 30mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml of IL-4 and/orIL-13 antibody in a quantity of 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7ml, 8 ml, 9 ml, 10 ml, 15 ml or 20 ml, for example.

An article of manufacture containing materials useful for the treatmentof the disorders described above is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for diagnosing,preventing or treating a IL-4 and/or IL-13 mediated condition or diseaseand may have a sterile access port (for example, the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The label on or associated with thecontainer indicates that the composition is used for treating thecondition of choice. The article of manufacture may further comprise asecond container comprising a pharmaceutically acceptable buffer, suchas phosphate-buffered saline, Ringer's solution and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including buffers, diluents, filters, needles, syringesand package inserts with instructions for use.

The invention now will be exemplified for the benefit of the artisan bythe following non-limiting examples that depict some of the embodimentsby and in which the instant invention can be practiced.

EXAMPLES Example 1 Sequencing of the Fv Domain of Mouse Anti-Human IL-13Monoclonal Antibody Clone B-B13

The reagent used in the method below was mouse anti-IL-13 monoclonalantibody clone B-B13 purchased from Cell Sciences, Inc. (Canton, Mass.,USA). Cell Sciences is the US distributor of Diaclone (Besançon,France), which manufactured the antibody B-B13.

The amino acid sequence of anti-IL-13 monoclonal antibody Clone B-B13was determined by a combination of Edman N-terminal sequencing and massspectrometric analysis. The antibody was subjected to the followingdifferent approaches in order to generate polypeptide or peptidefragments, and these were fractionated by different approaches in orderto prepare samples that were subsequently subjected to Edman N-terminalsequencing, and Liquid Chromatography/Mass Spectrometry/MassSpectrometry (LC-MS/MS) analysis with associated protein sequencedatabase peptide matching

SDS-Page of the antibody, either untreated or treated with pyrogluaminopeptidase, to separate the heavy and light chains, followed by blottingto polyvinylidene fluoride (PVDF) membrane and Edman N-terminalsequencing of the bands.

Limited partial proteolysis with specific proteases of the antibodyfollowed by SDS-Page and blotting to PVDF membrane and Edman N-terminalsequencing of the bands.

Limited partial chemical cleavage of the whole antibody, or heavy andlight chain SDS-Page gel bands, followed by SDS-Page and blotting toPVDF membrane and Edman N-terminal sequencing of bands.

Proteolysis of the whole antibody or heavy and light chain SDS-Page gelbands with specific proteases and LC/MS/MS analysis.

Proteolysis of heavy and light chain SDS-Page gel bands with specificproteases followed by reverse phase high pressure chromatographyfractionation (rp-hplc), and subsequent Edman N-terminal sequencing andLC/MS/MS analysis of fractions.

Limited proteolysis of the antibody with the protease papain,fractionation of the Fd (VH-CH1 fragment of the antibody heavy chain)gel band by SDS-Page, proteolysis with specific proteases, reverse phasehigh performance liquid chromatograph (rp-hplc), and subsequent EdmanN-terminal sequencing and LC/MS/MS analysis of fractions.

Example 2 Sequencing of the Fv Domain of Mouse Anti-Human IL-4Monoclonal Antibody Clone 8D4-8

The reagent mouse anti-IL-4 monoclonal antibody clone 8D4-8 waspurchased from Biozol diagnostica Vertrieb GmbH (Eching, Germany).Biozol is the German distributor of BioLegend (San Diego, Calif., USA)which manufactured the antibody 8D4-8.

The amino acid sequence of a mouse monoclonal anti-IL-4 antibody (clone8D4-8) was determined by a combination of Edman sequencing and massspectrometry (Pham et al., 2006, Anal. Biochem. 352: 77-86; Roberts etal., 2005, Anal. Chem. 67: 3613-25). Briefly, the antibody was firstseparated into light and heavy chains and then each chain was cleaved bysequence specific proteases or chemically. Resulting peptides wereseparated by reverse phase chromatography and analyzed byMatrix-assisted laser desorption/ionization spectrometry (MALDI) and/orLC-MS/MS. Unique peptides as well as the intact heavy and light chainswere than subjected to Edman sequencing for unambiguous determination ofthe protein sequence.

Example 3 Humanization of the Fv Domain of Mouse Anti-Human IL-13Monoclonal Antibody Clone B-B13

The humanization protocol described hereinabove was used to humanize theB-B13 clone. Six humanized versions were suggested which includemutations in the CDRs to address problematic residues (deamidation site,solvent exposed methionine, acide labile positions).

The VL & VH sequences of B-B13 were blasted against the July 2007version of the Protein Data Bank (PDB). The most similar light and heavychain amino acid sequences were retrieved. The closest homologue for thevariable light chain was found to be 1EGJ. The closest homologue for theheavy chain was found to be 1FNS. The structures 1EGJ & 1FNS were usedto build up a homology model of the variable domains which wassubsequently energy minimized using the standard procedure implementedin Molecular Operating Environment (MOE). MOE is a comprehensive suiteof softwares for computer assisted drug design distributed by theChemical Computing group. A molecular dynamic (MD) calculation of a 3Dhomology model of B-B13 was subsequently performed for 1.7 nanosecondsin Generalized Born implicit solvent. The resulting 1,700 snapshots fromthe MD trajectory were then used to calculate, for each B-B13amino-acid, the distribution of its root mean square deviations (rmsd)compared to a reference medoid position. A statistical test, comparingthe rmsd distribution of each amino-acid to the global rmsddistribution, is finally used to decide if the amino-acid is flexibleenough, as seen during the MD, to be considered as likely to interactwith B-cell receptors and responsible for activation of the immuneresponse. The flexible positions of the murine B-B13 variable regionwere compared to the corresponding positions in human antibody sequencesin the January 2007 version of the ImMunoGeneTics Database that has beendownloaded locally. Only those residues which display flexibilitygreater than three times the mean and a few flanking residues thatpreserve the 3D structures of these flexible residues were retained forthe search. The human antibody variable region with the most identicalflexible residues, with special considerations given to positions thatcome within 5.0 Å of a CDR, was chosen to replace the murine the B-B13antibody variable region flexible residues. A number of mutations in theCDRs were also included in the proposed versions to avoid problematicresidues. The following motifs of sequences were considered: Asp-Pro(acide labile bond), Asn-X-Ser/Thr (glycosylation), Asn-Gly/Ser/Thr(deamidation site in exposed area), Met (oxidation in exposed area). Theresulting humanized sequences were blasted for sequence similarityagainst UniProtKB/Swiss-Prot database providing confidence thatreasonable assumption has been made. It was found that all sequencesshow high degree of similarity to number of human antibodies. Inaddition none of the sequences contains any known B- or T-cell epitopelisted in the Immune Epitope Database and Analysis Resource (IEDBdatabase).

Three versions for the heavy chain (H1, H2, H3) and three versions weresuggested for the light chain (L1, L2, L3). The three versions of thelight chain are derived from CAA83271.1 (Genebank accession numberCAA83271). The L1 version has 4 mutations. The L2 version includes anadditional mutation to remove a DP site (Pro99) in CDR3. L3 incorporatestwo additional mutations located in the CDRs when compared with L2 whichare two presumed deamidation sites (N34Q, N96A). The H1, H2 and H3versions of the heavy chain are derived from CAC39364.1 (Genebankaccession number CAC39364). This template was not the top scoringtemplate but it was the highest scoring template which did not containsequence exhibiting high homology (>70%) with known immunogenicsequence. Version H1 contains 6 mutations and the H2 sequenceincorporates two additional mutations to address three deamidation sites(N60A, N73T, and N83T). The sequential numbering of amino acidy reflectstheir natural order within the protein (N-terminus to C-terminus). H3contains two additional mutations (Y100R & D106K) which were thought toimprove potency. Six combinations of VL and VH variants were recommendedfor generation of humanized antibodies: VL1×VH1, VL2×VH2, VL1×VH3,VL3×VH1, VL3×VH2 and VL3×VH3. As shown in Table 1, the amino acidchanges were made in humanized B-B13 VL and VH variants using there-surfacing technology set forth in the detailed description section ofthe instant application. The left column indicates the original aminoacids and their positions in the murine B-B13 mAb.

TABLE 1 Light Chain (Sequential numbering) (VL1) (VL2) (VL3) Asn1 AspAsp Asp Asn34 Asn Asn Gln Pro44 Ala Ala Ala Glu83 Gln Gln Gln Asp85 GluGlu Glu Asn96 Asn Asn Ala Pro99 Pro Ser Ser 4 mutations 5 mutations 7mutations Heavy Chain (VH1) (VH2) (VH3) Gln1 Glu Glu Glu Ser15 Gly GlyGly Gln16 Gly Gly Gly Asn60 Asn Ala Ala Ser61 Asp Asp Asp Asn73 Asn SerSer Lys81 Glu Glu Glu Asn83 Asn Thr Thr Gln86 Arg Arg Arg Tyr100 Tyr TyrArg Asp106 Asp Asp Lys 6 mutations 9 mutations 11 mutations

Example 4 Humanization of the Fv Domain of Mouse Anti-Human IL-4Monoclonal Antibody Clone 8D4-8

The humanization (resurfacing) technology described hereinabove was usedto humanize the 8D4-8 clone. Two humanized versions were prepared. Oneversion includes one mutation in the CDRs of the heavy chain which wasthought to address a problematic residue (exposed acide labilepositions).

The VL & VH sequences of 8D4-8 were blasted against the July 2007version of the PDB. The most similar light and heavy chain amino acidsequences were retrieved. The closest homologue for the variable lightchain is 1YDJ. The closest homologue for the heavy chain was found to be1IQW. The structures 1YDJ & 1IQW were used to build up a homology modelof the variable domains which was subsequently energy minimized usingthe standard procedure implemented in MOE. A molecular dynamic (MD)calculation of a 3D homology model of 8D4-8 was subsequently performedfor 1.7 nanoseconds in Generalized Born implicit solvent. The resulting1,700 snapshots from the MD trajectory were then used to calculate, foreach 8D4 amino-acid, the distribution of its root mean square deviations(rmsd) compared to a reference medoïd position. A statistical test,comparing the rmsd distribution of each amino-acid to the global rmsddistribution, is finally used to decide if the amino-acid is flexibleenough, as seen during the MD, to be considered as likely to interactwith B-cell receptors and responsible for activation of the immuneresponse. The flexible positions of the murine 8D4-8 variable regionwere compared to the corresponding positions in human antibody sequencesin the January 2007 version of the ImMunoGeneTics Database that has beendownloaded locally. Only those residues which display flexibilitygreater than three times the mean and a few flanking residues thatpreserve the 3D structures of these flexible residues were retained forthe search. The human antibody variable region with the most identicalflexible residues, with special considerations given to positions thatcome within 5.0 Å of a CDR, was chosen to replace the murine the 8D4-8antibody variable region flexible residues. Eventually, some additionalmutations were also made to avoid problematic residues. The followingmotifs of sequences were considered: Asp-Pro (acide labile bond),Asn-X-Ser/Thr (glycosylation), Asn-Gly/Ser/Thr (deamidation site inexposed area), Met (oxidation in exposed area). The only problematicresidue found was a DP site in the CDR2 of the heavy chain. Theresulting humanized sequences were blasted for sequence

Two versions for the heavy chain (H1, H2) and one version for the lightchain (L1) were suggested. The L1 version of the light chain is derivedfrom BAC01676.1 (Genebank accession number BAC01676). The L1 version has3 mutations. The H1 and H2 versions of the heavy chain are derived fromBAC02418.1 (Genebank accession number BAC02418). Version H1 contains 9mutations and the H2 version includes an additional mutation to remove aDP site (Pro53) in CDR2. Two combinations, VL1×VH1 and VL1×VH2, wereprepared.

Table 2 shows the amino acid changes that were made in humanized 8D4-8VL and VH variants using the humanization (re-surfacing) technology. Theleft column indicates the original amino acids and their positions inthe murine 8D4-8 mAb.

TABLE 2 Light Chain (Sequential numbering) (VL1) Asn5 Thr Leu15 ValSer39 Lys 3 mutations HeavyChain (VH1) (VH2) Gln10 Glu Glu Arg13 Lys LysThr16 Ala Ala Pro53 Pro Ala Lys65 Gln Gln Asp66 Gly Gly Arg74 Glu GluSer76 Thr Thr Leu93 Val Val Thr118 Leu Leu 9 mutations 10 mutations

Example 5 Cloning and Generation of Chimeric Anti-IL-13 Clone B-B13Monoclonal Antibody, a Chimeric Anti-IL-4 Clone 8D4-8 MonoclonalAntibody and Humanized Variants

Amino acid sequences of the variable heavy and light chains of theanti-IL-13 clone B-B13 and the anti-IL-4 clone 8D4-8 were backtranslatedinto nucleotide sequence and generated respectively using a modifiedprotocol of the overlap extension PCR (OE-PCR) described by Young L. andDong Q. (Nucl. Acids Res. (2004), 32(7), e59). PCR products were clonedinto the pCR®4-TOPO using the Invitrogen TOPO TA cloning kit (Cat#45-0641) and sequenced using M13 forward and M13 reverse primers. Thevariable domains were fused together to the constant heavy (IGHG1,Genebank accession number Q569F4) or light chain (IGKC) Genebankaccession number Q502W4) respectively, digested with NheI and HindIIIand each ligated into the NheI/HindIII sites of the episomal expressionvector pXL, an analogon of the pTT vector described by Durocher et al.(2002), Nucl. Acids Res. 30(2), E9, creating the plasmids for themammalian expression of the chimeric B-B13-heavy and light chains andthe chimeric 8D4-8 heavy and light chains.

The expression clones encoding the humanized variants of the anti-IL-13clone B-B13 and the anti-IL-4 clone 8D4-8 were also syntheticallygenerated by overlap extension PCR (OE-PCR), based on the proposed aminoacid exchanges of the original sequences.

The expression plasmids encoding the heavy and light chain of theantibody were propagated in E. coli DH5a. Plasmids used for transfectionwere prepared from E. coli using the Qiagen EndoFree Plasmid Mega Kit.

For transfection HEK293FreeStyle cells (Invitrogen) were seeded at 3×10⁵cells/mL in 100 mL volume of serum-free FreeStyle medium (Invitrogen) ina 500 mL shaker flask. Cells were cultured in a 37° C. incubator with ahumidified atmosphere of 8% CO₂, on an orbital shaker platform rotatingat 110 rpm.

Three days post-seeding viable and total cell were determined with aCASY electronic cell counter (Schärfe System GmbH). Cells with viabilitygreater than 90% were used for transfection at a cell density of1−1.5×10⁶ cells/mL. 100 mL cells were transfected in a 500 mL shakerflask with a mix of heavy and light chain expression plasmids (5×10⁻⁷μgDNA/cell) using FugeneHD (Roche) at a DNA:FugeneHD ratio of 2:7, atconditions described by the manufacturer. Transfected cells werecultured for 7 days in a 37° C. incubator (8% CO₂) on an orbital shakerplatform rotating at 110 rpm.

A Nunc F96-MaxiSorp-Immuno plate was coated with goat anti-Human IgG (Fcspecific) [NatuTcc A80-104A]. The antibody was diluted to 10 ug/ml incarbonate coating buffer (50 mM sodium carbonate pH 9.6) and dispensedat 50 uL per well. The plate was sealed with adhesive tape, and storedovernight at 4 C. The plate was washed 3 times with Wash buffer (PBS pH7.4 0.1% Tween20). 150 uL of blocking solution (1% BSA/PBS) wasdispensed into each well to cover the plate. After 1 hour at RT theplate was washed 3 times with Wash buffer. 100 uL of sample or standards(in a range from 1500 ng/ml to 120 ng/ml) were added and let sit for 1hour at RT. The plate was washed 3 times with Wash buffer. 100 uL ofgoat anti-Human IgG-FC-HRP conjugate [NatuTec A80-104P-60] diluted1:10.000 were added using incubation solution (0.1% BSA, PBS pH 7.4,0.05% Tween20). After 1 hour incubation at RT, the plate was washed 3times with Wash buffer. 100 uL of ABTS substrate (10 mg ABTS tablet(Pierce 34026) in ml of 0.1 M Na₂HPO₄, 0.05 M citric acid solution, pH5.0. Addition of 10 uL of 30% H₂O₂/10 ml Subtrate buffer prior to use)were dispensed to each well, allow the color to develop. After the colorhas developed (approximately 10 to 15 minutes), 50 uL of 1% SDS Solutionwere added to stop the reaction. The plate was read at A405.

Proteins were purified by affinity chromatography on Protein A (HiTrap™Protein A HP Columns, GE Life Sciences). After elution from the columnwith 100 mM acetate buffer with 100 mM NaCl pH 3.5, the monoclonalantibodies were formulated in PBS and 0.22 μm filtered. Proteinconcentration was determined by measurement of absorbance at 280 nm.Each batch was analyzed using a Protein 200 Plus LabChip kit on theAgilent 2100 bioanalyzer under reducing and non-reducing conditions todetermine the purity and the molecular weight of each subunit and of themonomer.

Example 6 Characterization of Humanized Anti-IL-13 Clone B-B13 Variantsand Humanized Anti-IL-4 Clone 8D4-8 Variants

The reagents recombinant human IL-13 and IL-4 were purchased fromChemicon (USA). The Biacore kinetic analysis was performed as follows.

Surface plasmon resonance technology on a Biacore 3000 (GE Healthcare)was used for detailed kinetic characterisation of purified antibodies. Acapture assay using a species specific antibody (e.g. human-Fc specificMAB 1302, Chemicon) for capture and orientation of the investigatedantibodies was used. The capture antibody was immobilied via primaryamine groups (10000 RU) on a research grade CM5 chip (GE Life Sciences)using standard procedures. The analysed antibody was captured with anadjusted RU value that would result in maximal analyte bindung of 30 RUat a flow rate of 10 μl/min. Binding kinetics were measured againstrecombinant human IL-4 and IL-13 over a concentration range between 0 to50 nM in HBS EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005%Surfactant P20) at a flow rate of 30 μl/min. Chip surfaces wereregenerated with 10 mM glycine pH2.5. Kinetic parameters were analysedand calculated in the BIAevaluation program package using a flow cellwithout captured antibody as reference. To investigate additive bindingof both antigens, a wizard-driven co-inject method has been applied inwhich one antigen was injected immediately followed by the antigen mixof IL-13/IL-4.

The antibodies of the present invention were measured for biologicalactivity by measuring the inhibition of IL-4 or IL-13 mediated cellproliferation in TF-1 cells. Briefly, Applicants used IL-4 or IL-13 tostimulate the growth of TF-1 cells. TF-1 is a cell line that isdependent on cytokines for growth and responds to many cytokinesincluding IL-4 and IL-13. The induced growth (compared to baselineconditions in the absence of cytokine) represents the biologicalactivity of IL-4 or IL-13. Anti-IL-4, anti-IL-13 and bispecificanti-IL-4/IL-13 antibodies were shown to block IL-4 or IL-13 inducedTF-1 cell growth. In addition, the bispecific anti-IL-4/IL-13 antibodieswere shown to block TF-1 cell proliferation induced by combined IL-4 andIL-13 stimulation. The blocking effect was measured in a dose-dependentmanner to generate IC50 (antibody concentration at 50% inhibition) asthe antibody neutralization potency against its target, i.e., IL-4 orIL-13. Details of the methods employed are described in more detailbelow.

TF-1 cells (ATCC, CRL-2003) were maintained in complete medium (DMEMwith high glucose, 25 mM Hepes buffer and glutamine, 10% FBS, 1×P/S, 1mM sodium pyruvate) containing freshly added hGM-CSF at finalconcentration of 4 ng/ml. 24 hrs before IL-13 (15 ng/ml) or IL-4 (1ng/ml) treatment. Cells were seeded in 96-well plates at 0.05×10⁶/ml incomplete medium without hGM-CSF. Serial dilutions of antibody with thecorresponding cytokine were pre-incubated for 30 minutes at 37° C.before adding to cells. Cells were cultured for 72 hours (37° C., 5%CO2). MTS/PMS solution of cellTiter 96 Aqueous was added. The cells werethen incubated for 3 hours. After that period, absorbance at 490 nmusing a plate reader was recorded. 1050 values were calculated usingSpeed software.

The binding kinetics and neutralization activity of humanized B-B13variants are shown in Table 3. (nt, not tested).

TABLE 3 on-rate off-rate KD IC50 antibody (M⁻¹ × S⁻¹) (S⁻¹) (M) (M)Murine B-B13 8.64E+05 3.73E−04 5.63E−10 Nt chB-B13 WT 1.76E+06 4.61E−042.61E−10 7.4E−9 huB-B13 VL1 × VH1 1.74E+06 6.91E−04 3.96E−10 1.57E−8 huB-B13 VL1 × VH3 1.93E+06 3.95E−04 2.05E−10 Nt huB-B13 VL2 × VH21.13E+06 1.77E−04 1.57E−10 Nt huB-B13 VL3 × VH1 1.93E+06 3.33E−041.72E−10 5.2E−9 huB-B13 VL3 × VH2 2.55E+06 1.12E−04 4.39E−11 3.2E−9huB-B13 VL3 × VH3 2.14E+06 4.05E−04 1.89E−10 Nt

One humanized B-B13 variant, huB-B13 VL3×VH2, has significantly higheraffinity compared with the original murine B-B13 (13 fold) and chimericB-B13 (6 fold). The improved affinity may lead to increased potency andefficacy when these humanized anti-IL-13 antibodies are used to treatasthma patients. In addition, the humanized antibodies may have reducedimmunogenicity compared with the murine antibody or the chimericantibody when used in man.

The binding kinetics and neutralization activity of humanized 8D4-8variants are shown in Table 4.

TABLE 4 on-rate off-rate KD IC50 antibody (M⁻¹ × S⁻¹) (S⁻¹) (M) (M)murine 8D4-8 5.57E+06 2.17E−04 3.77E−11 9.7E−11 ch8D4-8 WT 2.49E+071.95E−04 7.83E−12 8.4E−11 Hu8D4-8 4.72E+07 1.55E−04 3.29E−12 4.1E−11 VL1× VH1 Hu8D4-8 2.57E+07 3.48E−04 1.39E−11 1.35E−10  VL1 × VH2

One humanized 8D4-8 variant, hu8D4-8 VL1×VH1 has significantly higheraffinity compared with the original murine 8D4-8 (11 fold) and chimeric8D4-8 (2 fold). The improved affinity may lead to increased potency andefficacy when this humanized anti-IL-4 antibody is used to treat asthmapatients. In addition, the humanized antibody may have reducedimmunogenicity compared with the murine antibody or the chimericantibody when used in man.

Example 7 Cloning and Generation of Humanized Anti-IL-4/IL-13 BispecificAntibodies

The format used for the expression of bispecific antibodies (BsAb) is anIgG variant of the dual domain double head format described in U.S. Pat.No. 5,989,830. In this format an IgG molecule is elongated at itsN-terminus on the corresponding heavy and light chains, by an additionalvariable domain of a second antibody. Thus, the resulting IgG moleculeis a heterotetramer composed of two heavy and two light chains. Theheavy chains consist of two variables heavy domains (VH1-VH2) derivingfrom two different antibodies joined together by a linker composed often amino acids (G4S)₂ and fused to the IgG4 constant domain. The lightchains consist of two variables light domains (VL1-VL2) deriving fromtwo different antibodies joined together by a linker composed of tenamino acids (G4S)₂ and fused to the constant kappa region.

Sequences for the variable heavy and light domains of the 8D4-8 variantswere generated by PCR introducing a BamHI restriction site (GGA TCC) attheir respective 5′-ends encoding a part of the (G4S)₂-(GGA TCC)-8D4-8.The 3′ sequence of the VH of the 8D4-8 humanized variants ended with anApaI restriction site (encoding the first amino acids of the CH1 domain)for a later fusion to the IGHG4 sequence (Q569F4, with deletion of theterminal Lys and a double mutation S241P and L248E). The 3′-end of theVL8D4-8 ended with a BsiWI restriction site encoding the first two aminoacids of the constant kappa chain for a later fusion to IGKC (Gene BankAccession Number Q502W4).

Sequences for the variable heavy and light domains of the B-B13 variantswere generated by PCR introducing a BamHI restriction site at theirrespective 3′-ends encoding a part of the (G4S)₂—(B-B13)-(GGA GGC GGAGGG TCC GGA GGC GGA GGA TCC (SEQ ID NO: 7)). Both sequences for the VHand VL of the B-B13 variants were generated with a NheI restriction siteat their respective 5′-ends, followed by an ATG start codon and a leaderpeptide encoding sequence.

The VH of B-B13 and 8D4-8 were fused together through their BamHI siteswithin the (G4S)₂ linker. The VL of B-B13 and 8D4-8 were fused to eachother through their BamHI sites within the (G4S)₂ linker. Hence thetandems of heavy and the light chains generated had the followingcomposition.

Bispecific antibody heavy chain: NheI-Leader peptide-VH-B-B13-(G4S)₂-VH8D4-8-ApaI.

Bispecific antibody light chain: NheI-Leader peptide-VL-B-B13-(G4S)₂-VL8D4-8-BsiWI.

All intermediate PCR fragments were cloned into the pCR® 4-TOPO usingthe Invitrogen TOPO TA cloning kit (Cat #: 45-0641) and sequenced usingM13 forward and M13 reverse primers.

After sequence validation the heavy chain tandems were fused throughtheir ApaI site to the IGHG4 sequence and the variable light chaintandems were fused through their BsiWI site to IGKC. The created dualdomain heavy chain and light chain were digested with NheI and HindIIIand each ligated into the NheI/HindIII sites of the episomal expressionvector pXL, creating the plasmids for mammalian expression of theTBTI-heavy and light chains respectively.

Four humanized bispecific anti-IL-4/anti-IL-13 constructs were generatedbased on the following combinations of humanized VH and VL versions ofB-B13 and 8D4-8 as shown in Table 5.

TABLE 5 Bispecific anti-IL-4/anti-IL-13 Ab Anti-IL-13 Fv Anti-IL-4 FvhuTBTI3_1_1 B-B13 VL3 × VH2 8D4-8 VL1 × VH2 huTBTI3_2_1 B-B13 VL3 × VH28D4-8 VL1 × VH1 huTBTI3_1_2 B-B13 VL2 × VH2 8D4-8 VL1 × VH2 huTBTI3_2_2B-B13 VL2 × VH2 8D4-8 VL1 × VH1

Example 8 Characterization of the Humanized Bispecific Antibodies

Binding and neutralization activity assays were performed as describedin the previous Examples.

Table 6 depicts the binding kinetics of four humanized anti-IL-4/IL-13antibody variants. All four constructs of bispecific antibodies binds toIL-4 and IL-13 with high affinities.

TABLE 6 IL-13 affinity IL-4 affinity on-rate off-rate KD On-rateoff-rate KD BsAB (M⁻¹ × S⁻¹) (S⁻¹) (M) (M⁻¹ × S⁻¹) (S⁻¹) (M) huTBTI3-1_12.27E+06 1.70E−04 7.47E−11 2.55E+06 3.78E−04 1.48E−10 huTBTI3-2_12.17E+06 1.69E−04 7.80E−11 4.00E+06 1.39E−04 3.47E−11 huTBTI3-1_28.50E+05 1.64E−04 1.93E−10 2.23E+06 3.08E−04 1.38E−10 huTBTI3-2_28.20E+05 2.12E−04 2.59E−10 3.96E+06 1.32E−04 3.32E−11

The neutralization activity of humanized anti-IL-4/IL-13 bispecificantibody variants is summarized in Table 7. Both huTBTI3-1_(—)1 andhuTBTI3-2_(—)1 completely neutralized IL-13 or IL-4 induced TF-1 cellproliferation with IC50 shown below.

TABLE 7 Antibody IC50 (nM) in IL-13 assay IC50 (nM) in IL-4 assayhuTBTI3-1_1 3.7 1.7 huTBTI3-2_1 4.1 0.32

It is well known that a mutant IL-13 allele is linked in high frequencywith asthma (Heinzmann A. et al., 2000, Hum Mol Genet. 9, 4, p 549-559).Therefore, the binding kinetics of the bispecific antibodies to themutant IL-13 protein (human IL-13 R112Q variant, PeproTech, Rocky Hill,N.J., USA) was studied. The results indicated that the huTBTI3-1_(—)1and huTBTI3-2_(—)1 bound to the IL-13 variant similarly to the wild typeIL-13.

Table 8 shows the binding kinetics of humanized anti-IL-4/IL-13molecules to mutant IL-13 protein.

TABLE 8 IL13 variant affinity on-rate off-rate KD BsAB (M⁻¹ × S⁻¹) (S⁻¹)(M) huTBTI3-1_1 9.74E+05 1.18E−04 1.21E−10 huTBTI3-2_1 9.48E+05 2.00E−042.11E−10

1-49. (canceled) 50: A method of treating an IL-4 and/or IL-13 mediateddisease in a mammal comprising the step of administering to the mammal atherapeutically effective amount of a bispecific antibody or bispecificantibody fragment thereof that specifically binds to IL-13 and IL-4,wherein the bispecific antibody or bispecific antibody fragment thereofcomprises a first pair of polypeptides and a second pair ofpolypeptides; wherein the first pair of polypeptides comprise an outer(N-terminal) variable light chain domain linked to an inner (C-terminal)variable light chain domain which is linked to a constant light chaindomain (CL), and the second pair of polypeptides comprise an outer(N-terminal) variable heavy chain domain linked to an inner (C-terminal)variable heavy chain domain which is linked to a constant heavy chaindomain (CH1); and wherein: (a) the outer (N-terminal) variable lightchain domain comprises the amino acid sequence of SEQ ID NO: 1, theinner (C-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 3, the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequence of SEQ ID NO: 2, and the inner(C-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 4; or (b) the outer (N-terminal) variable lightchain domain comprises the amino acid sequence of SEQ ID NO: 1, theinner (C-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 3, the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequence of SEQ ID NO: 2, and the inner(C-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 5; (c) the outer (N-terminal) variable lightchain domain comprises the amino acid sequence of SEQ ID NO: 3, theinner (C-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 1, the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequence of SEQ ID NO: 4, and the inner(C-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 2; (d) the outer (N-terminal) variable lightchain domain comprises the amino acid sequence of SEQ ID NO: 3, theinner (C-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 1, the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequence of SEQ ID NO: 5, and the inner(C-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 2; (e) the outer (N-terminal) variable lightchain domain comprises the amino acid sequences RASESVDSYGQSYMH (SEQ IDNO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10), the inner(C-terminal) variable light chain domain comprises the amino acidsequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), andQQAHSYPFT (SEQ ID NO: 16), the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11):DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13), and the inner(C-terminal) variable heavy chain domain comprises the amino acidsequences GYSFTSYWIH (SEQ ID NO: 17), IDPSDGETR (SEQ ID NO: 18), andLKEYGNYDSFYFDV (SEQ ID NO: 19); (f) the outer (N-terminal) variablelight chain domain comprises the amino acid sequences RASESVDSYGQSYMH(SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10),the inner (C-terminal) variable light chain domain comprises the aminoacid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15),and QQAHSYPFT (SEQ ID NO: 16), the outer (N-terminal) variable heavychain domain comprises the amino acid sequences GFSLTDSSIN (SEQ ID NO:11): DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13), and theinner (C-terminal) variable heavy chain domain comprises the amino acidsequences GYSFTSYWIH (SEQ ID NO: 20), IDASDGETR (SEQ ID NO: 21), andLKEYGNYDSFYFDV (SEQ ID NO: 22); (g) the outer (N-terminal) variablelight chain domain comprises the amino acid sequences HASQNIDVWLS (SEQID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), theinner (C-terminal) variable light chain domain comprises the amino acidsequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), andQQNAEDSRT (SEQ ID NO: 10), the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17),IDPSDGETR (SEQ ID NO: 18), and LKEYGNYDSFYFDV (SEQ ID NO: 19), and theinner (C-terminal) variable heavy chain domain comprises the amino acidsequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), andDGYFPYAMDF (SEQ ID NO: 13); or (h) the outer (N-terminal) variable lightchain domain comprises the amino acid sequences HASQNIDVWLS (SEQ ID NO:14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), the inner(C-terminal) variable light chain domain comprises the amino acidsequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), andQQNAEDSRT (SEQ ID NO: 10), the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequences GYSFTSYWIH (SEQ ID NO: 20),IDASDGETR (SEQ ID NO: 21), LKEYGNYDSFYFDV (SEQ ID NO: 22), and the inner(C-terminal) variable heavy chain domain comprises the amino acidsequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), andDGYFPYAMDF (SEQ ID NO: 13).
 51. (canceled) 52: The method of claim 50,wherein the IL-4 and/or IL-13 mediated disease is an allergic disease,cancer, asthma, a disease associated with abnormal production of IL-4and/or IL-13, an autoimmune disease, scleroderma, or idiopathicpulmonary fibrosis. 53: A method of suppressing a TH-2 mediated responsein a mammal comprising the step of administering to the mammal atherapeutically effective amount of a bispecific antibody or bispecificantibody fragment thereof that specifically binds to IL-13 and IL-4,wherein the bispecific antibody or bispecific antibody fragment thereofcomprises a first pair of polypeptides and a second pair ofpolypeptides; wherein the first pair of polypeptides comprise an outer(N-terminal) variable light chain domain linked to an inner (C-terminal)variable light chain domain which is linked to a constant light chaindomain (CL), and the second pair of polypeptides comprise an outer(N-terminal) variable heavy chain domain linked to an inner (C-terminal)variable heavy chain domain which is linked to a constant heavy chaindomain (CH1); and wherein: (a) the outer (N-terminal) variable lightchain domain comprises the amino acid sequence of SEQ ID NO: 1, theinner (C-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 3, the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequence of SEQ ID NO: 2, and the inner(C-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 4; or (b) the outer (N-terminal) variable lightchain domain comprises the amino acid sequence of SEQ ID NO: 1, theinner (C-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 3, the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequence of SEQ ID NO: 2, and the inner(C-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 5; (c) the outer (N-terminal) variable lightchain domain comprises the amino acid sequence of SEQ ID NO: 3, theinner (C-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 1, the outer domain (N-terminal) variable heavychain domain comprises the amino acid sequence of SEQ ID NO: 4, and theinner (C-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 2; (d) the outer (N-terminal) variable lightchain domain comprises the amino acid sequence of SEQ ID NO: 3, theinner (C-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 1, the outer domain (N-terminal) variable heavychain domain comprises the amino acid sequence of SEQ ID NO: 5, and theinner (C-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 2; (e) the outer (N-terminal) variable lightchain domain comprises the amino acid sequences RASESVDSYGQSYMH (SEQ IDNO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10), the inner(C-terminal) variable light chain domain comprises the amino acidsequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), andQQAHSYPFT (SEQ ID NO: 16), the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11),DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13), and the inner(C-terminal) variable heavy chain domain comprises the amino acidsequences GYSFTSYWIH (SEQ ID NO: 17), IDPSDGETR (SEQ ID NO: 18), andLKEYGNYDSFYFDV (SEQ ID NO: 19); (f) the outer (N-terminal) variablelight chain domain comprises the amino acid sequences RASESVDSYGQSYMH(SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10),the inner (C-terminal) variable light chain domain comprises the aminoacid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15),and QQAHSYPFT (SEQ ID NO: 16), the outer (N-terminal) variable heavychain domain comprises the amino acid sequences GFSLTDSSIN (SEQ ID NO:11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13), the inner(C-terminal) variable heavy chain domain comprises the amino acidsequences GYSFTSYWIH (SEQ ID NO: 20), IDASDGETR (SEQ ID NO: 21), andLKEYGNYDSFYFDV (SEQ ID NO: 22); (g) the outer (N-terminal) variablelight chain domain comprises the amino acid sequences HASQNIDVWLS (SEQID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), theinner (C-terminal) variable light chain domain comprises the amino acidsequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), andQQNAEDSRT (SEQ ID NO: 10), the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17),IDPSDGETR (SEQ ID NO: 18), and LKEYGNYDSFYFDV (SEQ ID NO: 19), and theinner (C-terminal) variable heavy chain domain comprises the amino acidsequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), andDGYFPYAMDF (SEQ ID NO: 13); or (h) the outer (N-terminal) variable lightchain domain comprises the amino acid sequences HASQNIDVWLS (SEQ ID NO:14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), the inner(C-terminal) variable light chain domain comprises the amino acidsequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), andQQNAEDSRT (SEQ ID NO: 10), the outer (N-terminal) variable heavy chaindomain comprises the amino acid sequences GYSFTSYWIH (SEQ ID NO: 20),IDASDGETR (SEQ ID NO: 21), and LKEYGNYDSFYFDV (SEQ ID NO: 22), and theinner (C-terminal) variable heavy chain domain comprises the amino acidsequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), andDGYFPYAMDF (SEQ ID NO: 13).
 54. (canceled) 55: A nucleic acid moleculeencoding a bispecific antibody or bispecific antibody fragment thereofthat specifically binds to IL-13 and IL-4, wherein the bispecificantibody or bispecific antibody fragment thereof comprises a first pairof polypeptides and a second pair of polypeptides; wherein the firstpair of polypeptides comprise an outer (N-terminal) variable light chaindomain linked to an inner (C-terminal) variable light chain domain whichis linked to a constant light chain domain (CL), and the second pair ofpolypeptides comprise an outer (N-terminal) variable heavy chain domainlinked to an inner (C-terminal) variable heavy chain domain which islinked to a constant heavy chain domain (CH1); and wherein: (a) theouter (N-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 1, the inner (C-terminal) variable light chaindomain comprises the amino acid sequence of SEQ ID NO: 3, the outer(N-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 2, and the inner (C-terminal) variable heavychain domain comprises the amino acid sequence of SEQ ID NO: 4; or (b)the outer (N-terminal) variable light chain domain comprises the aminoacid sequence of SEQ ID NO: 1, the inner (C-terminal) variable lightchain domain comprises the amino acid sequence of SEQ ID NO: 3, theouter (N-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 2, and the inner (C-terminal) variable heavychain domain comprises the amino acid sequence of SEQ ID NO: 5; (c) theouter (N-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 3, the inner (C-terminal) variable light chaindomain comprises the amino acid sequence of SEQ ID NO: 1, the outerdomain (N-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 4, and the inner (C-terminal) variable heavychain domain comprises the amino acid sequence of SEQ ID NO: 2; (d) theouter (N-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 3, the inner (C-terminal) variable light chaindomain comprises the amino acid sequence of SEQ ID NO: 1, the outer(N-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 5, and the inner (C-terminal) variable heavychain domain comprises the amino acid sequence of SEQ ID NO: 2; (e) theouter (N-terminal) variable light chain domain comprises the amino acidsequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), andQQNAEDSRT (SEQ ID NO: 10), the inner (C-terminal) variable light chaindomain comprises the amino acid sequences HASQNIDVWLS (SEQ ID NO: 14),KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), the outer(N-terminal) variable heavy chain domain comprises the amino acidsequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), andDGYFPYAMDF (SEQ ID NO: 13), and the inner (C-terminal) variable heavychain domain comprises the amino acid sequences GYSFTSYWIH (SEQ ID NO:17), IDPSDGETR (SEQ ID NO: 18), and LKEYGNYDSFYFDV (SEQ ID NO: 19); (f)the outer (N-terminal) variable light chain domain comprises the aminoacid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9),and QQNAEDSRT (SEQ ID NO: 10), the inner (C-terminal) variable lightchain domain comprises the amino acid sequences HASQNIDVWLS (SEQ ID NO:14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), the outer(N-terminal) variable heavy chain domain comprises the amino acidsequences GFSLTDSSIN (SEQ ID NO: 11) DGRID (SEQ ID NO: 12), andDGYFPYAMDF (SEQ ID NO: 13), and the inner (C-terminal) variable heavychain domain comprises the amino acid sequences GYSFTSYWIH (SEQ ID NO:20), IDASDGETR (SEQ ID NO: 21), and LKEYGNYDSFYFDV (SEQ ID NO: 22); (g)the outer (N-terminal) variable light chain domain comprises the aminoacid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15),and QQAHSYPFT (SEQ ID NO: 16), the inner (C-terminal) variable lightchain domain comprises the amino acid sequences RASESVDSYGQSYMH (SEQ IDNO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10), the outer(N-terminal) variable heavy chain domain comprises the amino acidsequences GYSFTSYWIH ID NO: 17), IDPSDGETR (SEQ ID NO: 18), andLKEYGNYDSFYFDV (SEQ ID NO: 19), and the inner (C-terminal) variableheavy chain domain comprises the amino acid sequences GFSLTDSSIN (SEQ IDNO: 11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13); or (h)the outer (N-terminal) variable light chain domain comprises the aminoacid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15),and QQAHSYPFT (SEQ ID NO: 16), the inner (C-terminal) variable lightchain domain comprises the amino acid sequences RASESVDSYGQSYMH (SEQ IDNO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10), the outer(N-terminal) variable heavy chain domain comprises the amino acidsequences GYSFTSYWIH (SEQ ID NO: 20), IDASDGETR (SEQ ID NO: 21), andLKEYGNYDSFYFDV (SEQ ID NO: 22), and the inner (C-terminal) variableheavy chain domain comprises the amino acid sequences GFSLTDSSIN (SEQ IDNO: 11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13). 56.(canceled) 57: A vector comprising the nucleic acid molecule of claim55. 58: A host cell comprising the vector of claim
 57. 59: A method ofmanufacturing a bispecific antibody or bispecific antibody fragmentthereof that specifically binds to IL-13 and IL-4, comprising expressingthe nucleic acid molecule of claim 55 in a suitable host cell. 60: Abispecific antibody or bispecific antibody fragment thereof thatspecifically binds to IL-13 and IL-4, wherein the bispecific antibody orbispecific antibody fragment thereof comprises a first pair ofpolypeptides and a second pair of polypeptides; wherein the first pairof polypeptides comprise an outer (N-terminal) variable light chaindomain linked to an inner (C-terminal) variable light chain domain whichis linked to a constant light chain domain (CL), and the second pair ofpolypeptides comprise an outer (N-terminal) variable heavy chain domainlinked to an inner (C-terminal) variable heavy chain domain which islinked to a constant heavy chain domain (CH1); and wherein: (a) theouter (N-terminal) variable light chain domain comprises the amino acidsequence of SEQ ID NO: 1, the inner (C-terminal) variable light chaindomain comprises the amino acid sequence of SEQ ID NO: 3, wherein afirst peptide linker links the amino acid sequence of SEQ ID NO: 1 tothe amino acid sequence of SEQ ID NO: 3, and wherein the first peptidelinker consists of the amino acid sequence of SEQ ID NO: 6; (b) theouter (N-terminal) variable heavy chain domain comprises the amino acidsequence of SEQ ID NO: 2, and the inner (C-terminal) variable heavychain domain comprises the amino acid sequence of SEQ ID NO: 4, whereina second peptide linker links the amino acid sequence of SEQ ID NO: 2 tothe amino acid sequence of SEQ ID NO: 4, and wherein the second peptidelinker consists of the amino acid sequence of SEQ ID NO: 6; and whereinthe bispecific antibody or bispecific antibody fragment thereof furthercomprises additional constant region domains, wherein the additionalconstant region domains consist of C_(H2) and C_(H3).